CN113163836A - Compositions and methods for promoting host defense and stimulating, expanding and/or resetting T cell banks - Google Patents

Compositions and methods for promoting host defense and stimulating, expanding and/or resetting T cell banks Download PDF

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CN113163836A
CN113163836A CN201980051348.4A CN201980051348A CN113163836A CN 113163836 A CN113163836 A CN 113163836A CN 201980051348 A CN201980051348 A CN 201980051348A CN 113163836 A CN113163836 A CN 113163836A
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composition
bifidobacterium
oligosaccharide
vitamin
threonine
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S·A·福雷瑟
B·亨利克
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Infinant Health Inc
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Evolve Biosystems Inc
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Abstract

Compositions comprising vitamin a, vitamin D, threonine, mammalian milk oligosaccharides and bifidobacteria, bifidobacterium infantis bifidobacterium cell wall components and their use in the treatment or prevention of disease, supportive therapy, including metabolic and immune diseases.

Description

Compositions and methods for promoting host defense and stimulating, expanding and/or resetting T cell banks
Technical Field
The invention described herein relates generally to compositions and methods for promoting host defense by modulating gut microbiome and/or biochemistry to improve barrier function and B or T immune cell pathways within the immune system. Compositions and methods for modulating immune cell function include various combinations that may include one or more of the following: vitamin a or a derivative thereof, a Bifidobacterium (Bifidobacterium) species, different forms of Bifidobacterium infantis (b.infarnatis) or a cell wall component thereof (whether the cell is viable or dead), vitamin D, threonine and/or oligosaccharides; when administered to the gut of an animal, particularly a human in need of stimulation of the naive or mature immune system, conditions such as immune immaturity, immune dysfunction or direct immune function stimulation will be ameliorated to improve specific immunotherapy.
Background
The gut microbiome and its function are increasingly considered to be a critical part of health and disease and are critical to the normal function of the immune system. The gastrointestinal tract or intestinal tract is exposed daily to a large number of antigens, including bacteria and food. Host defense is at multiple levels, including the physical barrier of the gut epithelium, the composition of the gut microbiome and the innate and acquired immune systems.
B cells are lymphocytes, which are part of the antigen recognition pathway leading to antibody production and are part of the adaptive immunity, while regulatory T (treg) cells are a specialized CD4+ T cell lineage that plays an important role in maintaining self-tolerance. Dysfunction of these cells may be associated with the development of various autoimmune and allergic diseases.
Retinoic acid is a vitamin a metabolite that regulates a variety of biological processes, including cell differentiation and proliferation. Recent studies have shown that retinoic acid also regulates the differentiation of T helper and Treg cells, and has been shown to maintain the stability of tregs in inflammatory conditions. Lui et al (2015)Cellular&Molecular Immunology 12：553-557。
An exhaustive list of over 1000 microorganisms in the human microbiome was studied, which concluded that most bacteria do not have the ability to stimulate tregs. It has been found that the ability to stimulate tregs is limited to 38 species in the Clostridia Class (Clostridia Class), which are capable of leading to robust Treg induction in the colon, followed by elevated IL-10 levels in the colon [ us patent application publication No. 2016/0193257 ]. In other experiments, polysaccharide a (PSA), a member of the class clostridia, produced by the PSA locus of Bacteroides fragilis (Bacteroides fragilis), or other synthetic zwitterionic polysaccharides, present on the outer surface of bacteria, have been used to stimulate tregs to treat, prevent or control inflammation and inflammatory disorders [ us patent publication 2016/0030464 and us patent publication 2014/0072534 ].
In a mouse study, bifidobacterium longum (b.longum) was found to reduce Peyer's patch gene expression of peptides associated with antigen presentation, TLR signaling and cytokine production, while increasing expression of genes associated with retinoic acid metabolism and induced regulatory T cells in an adult mouse allergy model. Bifidobacterium breve (b. breve) in infant mice had an effect on Foxp3+ regulatory T cells but no protective effect on respiratory or oral allergies [ Lyons et al (2010)Clinical and Experimental Allergy(40)：811-819]。
Disclosure of Invention
The present invention provides a composition for food or therapeutic use comprising a component selected from the group consisting of: vitamin A or its derivatives, Bifidobacterium infantis solute-binding protein, Bifidobacterium infantis exopolysaccharide fraction, oligosaccharide, bioavailable threonine and vitamin D.
The present invention provides a composition comprising vitamin a or a vitamin a derivative or metabolite thereof and an Oligosaccharide (OS).
The vitamin A may be retinol, retinal, retinoic acid, provitamin A carotenoids, or combinations thereof. The provitamin a carotenoid may be alpha-carotene, beta-carotene, gamma-carotene, lutein beta-cryptoxanthin, or combinations thereof. The microbial a-procarotene may be beta-carotene. The composition may include delivery of 1-10,000 international units of vitamin a per day. The composition may include delivery of 1-2,000 international units of vitamin a per day. The composition may comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μmol/l vitamin a. The composition may comprise about 1-100, 5-50, 25-75, 10-100, 30-60, or 75-100 μmol/l vitamin A.
In any of the preceding embodiments, the Oligosaccharide (OS) may comprise one or more oligosaccharides having 2-10 residues (DP2-10 oligosaccharides). The OS may be a Mammalian Milk Oligosaccharide (MMO). The Mammalian Milk Oligosaccharide (MMO) may include oligosaccharide molecules present in Human Milk Oligosaccharide (HMO), Bovine Milk Oligosaccharide (BMO), Bovine Colostrum Oligosaccharide (BCO), Goat Milk Oligosaccharide (GMO), or a combination thereof. Oligosaccharides may include carbohydrate polymers in mammalian milk that are not metabolized by any combination of digestive enzymes expressed by mammalian genes. Oligosaccharide compositions may include lacto-N-disaccharide (LNB), N-acetyllactosamine (lactosamine), lacto-N-trisaccharide, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), fucosyllactose (fucosyllactose, FL), lacto-N-fucopentose (LNFP), lacto-difucotetraose (LDFT), Sialyllactose (SL), disialyllacto-N-tetraose (DSLNT), 2 '-fucosyllactose (2FL), 3' -sialyllactosamine (3 '-sialyllactosamine, 3SLN), 3' -fucosyllactose (3FL), 3 '-sialyl-3-fucosyllactose (3S3FL), 3' -sialyllactose (3SL), 6 '-sialyllactosamine (6SLN), 6' -sialyllactose (6SL), one or more of Difucosyllactose (DFL), lacto-N-fucopentaose i (lnfpi), lacto-N-fucopentaose ii (lnfpii), lacto-N-fucopentaose iii (lnfpiii), lacto-N-fucopentaose v (lnfpv), sialyllacto-N-tetraose (SLNT), derivatives thereof, or combinations thereof. The oligosaccharides may include: (a) one or more type II oligosaccharide cores, wherein representative materials include lnnts; (b) one or more oligosaccharides comprising a type II core and GOS in a ratio of 1: 5 to 5: 1; (c) one or more oligosaccharides comprising a type II core and 2FL in a ratio of 1: 5 to 5: 1; (d) combinations of (a), (b), and/or (c); (e) one or more type I oligosaccharide cores, wherein representative materials include LNT, (f) one or more type I cores and GOS in a ratio of 1: 5 to 5: 1; (g) one or more type I cores and 2FL in a ratio of 1: 5 to 5: 1; and/or (h) any combination of (a) - (g) including type I and type II cores. Form I or form II may be isomeric with each other. Other type II cores include, but are not limited to, trifucosylated lacto-N-hexoses (TFLNH), LnNH, lacto-N-hexoses (LNH), lacto-N-fucopentasaccharide iii (lnfpiii), monofucosylated lacto-N-hexoses iii (mflnhiii), monofucosylated monosalivary lacto-N-hexoses (MFMSLNH).
In any of the preceding embodiments, the oligosaccharide may be from an animal, fungus, crustacean, insect, or plant. In some embodiments, the oligosaccharide may be a plant-derived oligosaccharide. The plant oligosaccharide can be from carrot, pea, broccoli, onion, tomato, pepper, rice, soybean, wheat, oat, bran, orange, cocoa, olive, apple, grape, beet, cabbage, corn, or combinations thereof. The plant oligosaccharide may be a pre-digested polysaccharide from orange peel, cocoa bean hull, olive pomace, tomato peel, grape pomace, corn silage or a mixture thereof. Plant derived oligosaccharides may be 2-10 sugar residues (DP2-DP10), 3-10 sugar residues (DP3-DP10), 5-10 sugar residues (DP5-DP10), or greater than DP 20. In some embodiments, the fungal, insect or crustacean polysaccharide may be predigested to produce oligosaccharides. In some embodiments, the chitin or chitosan is treated to produce fragments that may be N-acetylglucosamine or N-acetylgalactosamine (NAG), or the NAG polymer may be DP2-DP 20.
In any of the preceding embodiments, the oligosaccharide comprises galacto-oligosaccharide (GOS) or fructo-oligosaccharide (FOS) or xylo-oligosaccharide (XOS).
In any of the preceding embodiments, the Oligosaccharide (OS) may comprise a Human Milk Oligosaccharide (HMO) from any source.
In any of the preceding embodiments, the composition may provide an amount of total dietary intake of oligosaccharides of 0.001 to 100 g/day. The amount of oligosaccharide may be 1-20 g, 3-20 g, 5-10 g, 10-40 g per unit dose. The amount of oligosaccharide may be 10, 15, 20, 25, 30, 35, 40, 45 or 50 grams. In various embodiments, the total grams per day may be delivered in batches multiple times a day, or in bolus (bolus) administered once a day.
In any of the preceding embodiments, the composition may further comprise bifidobacteria. The Bifidobacterium may be Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium animalis subsp.animalis, Bifidobacterium animalis subsp.lactis, Bifidobacterium bifidum (b.bifidobacterium), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium catenulatum (Bifidobacterium catenulatum), Bifidobacterium longum subsp.infantis, Bifidobacterium pseudocatenulatum (b.pseudocatenulatum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), or a combination thereof. The composition may comprise activated bifidobacteria. The bifidobacterium longum may be bifidobacterium longum subsp. In a preferred embodiment, the bifidobacterium infantis has a functional H5 cluster. The bifidobacterium longum subspecies infantis may be an activated bifidobacterium longum subspecies infantis. Extracellular polysaccharides and solute-binding proteins on the cell surface of bifidobacterium infantis may be increased. The bifidobacterium may be bifidobacterium breve. The bifidobacterium breve may be activated bifidobacterium breve.
In any of the preceding embodiments, the composition may comprise bifidobacteria in an amount of 10-5000 million Colony Forming Units (CFU) per gram of composition. The composition may include bifidobacteria in an amount of 100-1000 million Colony Forming Units (CFU), 10-1 million, 100-50 million, or 50-200 million Colony Forming Units (CFU) per gram of the composition. The amount of bifidobacteria may be 100, 1000, 1, 10, 50, 150, 200, 250, 300, 350, 400, 450 or 500 million Colony Forming Units (CFU) per gram of composition. The amount of bifidobacteria may be 50-200 million Colony Forming Units (CFU) per gram of composition or 50-200 million colony forming units per gram of composition or 10-1 million colony forming units per gram of composition.
Any embodiment of the present invention may include, but is not limited to, increasing the bioavailability of threonine, N-acetyl threonine or gamma-glutamyl threonine in the intestine.
In some embodiments, the vitamin D status is monitored. In some embodiments, vitamin D is added to the oil formulation. In some embodiments, vitamin D and bifidobacteria are in the MCT oil composition, optionally together with vitamin a. In a preferred embodiment, the bifidobacterium is bifidobacterium infantis, which is optionally activated. In some embodiments, the total dietary intake of vitamin D is increased in a subject in need of treatment for any of the disorders described herein. In some embodiments, vitamin D is added to milk in the diet. Vitamin D can be in the form of drops, capsules or powders.
The composition may further comprise an isolated bifidobacterium infantis activated cell membrane comprising exopolysaccharides and/or solute-binding proteins. In some embodiments, the intact dead cells are delivered in a composition.
In any embodiment, the composition may be in the form of a dry powder or a dry powder suspended in an oil. The composition may be spray dried or freeze dried. The composition may be freeze-dried in the presence of a cryoprotectant.
In any of the preceding embodiments, the composition may further comprise a stabilizer. The stabilizing agent may be a flow agent. The stabilizer may be a cryoprotectant. The cryoprotectant is glucose, lactose, raffinose, sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethylene glycol, propylene glycol, ribitol, alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidone, taurine, mammalian lactooligosaccharide, polysaccharide or a combination thereof.
In any of the preceding embodiments, the composition may be administered in a food composition, such as mammalian milk, a mammalian milk-derived product, a mammalian donor milk, a human milk product, an infant formula, a milk substitute, an enteral nutritional product, and/or a meal substitute. OS may be administered as a powder, which may be in a sachet, stick pack, capsule, tablet, or may be administered as a liquid, such as in the form of a syrup, or may be suspended in other liquids, including non-aqueous solutions such as oils or gels or pastes. The non-bacterial composition may be in an aqueous solution. The aqueous solution may be sterile.
In any of the preceding embodiments, the composition may be formulated as a unit dose medicament.
In any of the above embodiments, the composition may be a pharmaceutical composition, a dietary supplement, a nutritional product, a food product, a probiotic and/or a prebiotic.
In any of the above embodiments, the composition may be formulated as a capsule, packet, sachet, food, lozenge, tablet, optionally effervescent tablet, enema, suppository, dry powder suspended in oil, chewable composition, syrup or gel.
In any of the above embodiments, the composition may further comprise an intact protein source or a breakdown product enriched in threonine, N-acetyl-threonine, gamma-glutamyl threonine, or a combination thereof.
The present invention also provides nutritional products comprising the compositions described herein. The nutritional product may be a food product, a dietary supplement, an infant formula or a pharmaceutical product.
Information for carrying out the methods described herein is further described in U.S. provisional patent application No. 62/558,349. Any of the methods described herein can reduce taste disturbances, the risk of development of autoimmune, inflammatory, or metabolic diseases in a mammal compared to a mammal with a metabolic disorder, such as, but not limited to, juvenile diabetes (type I), obesity, diabetes (type II) asthma, atopy, inflammatory bowel disease, celiac disease, food allergies, autism. It is expected that risk will be reduced by a statistically significant amount. For example, the risk may be reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
The methods described herein can increase the function of the immune system in a mammal, such as improving vaccine response and/or mucosal innate or adaptive immunity, and/or improving the production and transfer of secretory IgA in the gut of a mammal. It is expected that the response will improve by a statistically significant amount. For example, the reaction may be increased by 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
The methods described herein can increase the function of the immune system in a mammal, such as improving the effectiveness of immunotherapy, and/or improving the specificity and sensitivity of a particular immunotherapy. It is expected that the response will improve by a statistically significant amount. For example, the reaction may be increased by 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
The present invention also provides methods for preventing and/or treating autoimmune diseases comprising administering the compositions described herein.
In one embodiment, a method for elevating regulatory T cells (tregs) and/or B cells comprises administering retinoic acid or a source thereof, an Oligosaccharide (OS), and optionally, bifidobacterium to a subject. In other embodiments, vitamin a status is measured and a vitamin a diet is recommended as a supplement for treatment with OS and, optionally, by bifidobacteria suitably provided by the diet. In some embodiments, a treatment regimen may involve a sequence including different formulations comprising one or more of OS, vitamin a, and bifidobacteria for the initiation and maintenance phases.
In one embodiment, the method for preventing and/or treating an autoimmune disease comprises administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally bifidobacterium. In some embodiments, the status of vitamin a is monitored systemically or in a fecal sample to determine vitamin a availability and adjust the treatment accordingly.
In one embodiment, the method for preventing and/or treating allergy comprises administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally bifidobacterium.
In one embodiment, a method for increasing the efficiency of antigen recognition in an animal comprises administering to the subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally, bifidobacterium. The efficiency of gene therapy and/or vaccines can be improved in subjects in need thereof.
In one embodiment, the method for maintaining gut mucosal integrity during chemotherapy comprises administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, and an Oligosaccharide (OS), optionally, bifidobacterium.
In one embodiment, the method for preventing and/or treating an autoimmune disease comprises administering: (a) vitamin a or a vitamin a derivative or metabolite or source thereof; (b) oligosaccharides (OS); and (c) bifidobacteria.
In one embodiment, the method for preventing and/or treating allergy comprises administering: (a) vitamin a or a vitamin a derivative or metabolite or source thereof; (b) oligosaccharides (OS); and (c) bifidobacteria.
In one such method, the method for preserving intestinal barrier integrity during chemotherapy or radiation therapy comprises administering: (a) oligosaccharides (OS); (b) a bifidobacterium; and (c) threonine, N-acetyl threonine and/or gamma-glutamyl threonine rich proteins; and (d) optionally, vitamin a or a derivative thereof. In one embodiment, the method for maintaining gut mucosal integrity during chemotherapy comprises administering: (a) vitamin a or a vitamin a derivative or metabolite or source thereof; (b) oligosaccharides (OS); (c) a bifidobacterium; and (d) threonine and/or threonine, N-acetyl threonine and/or gamma-glutamyl threonine rich proteins.
In one embodiment, a method for stimulating regulatory t (treg) cells comprises administering: (a) oligosaccharides (OS); (b) a bifidobacterium; and (c) optionally, vitamin a or a derivative thereof.
In one embodiment, a method for stimulating mucin production comprises administering: (a) oligosaccharides (OS); (b) a bifidobacterium; and (c) threonine and/or threonine, N-acetyl threonine and/or gamma-glutamyl threonine rich proteins. In some embodiments, the composition of (a) or (b) or (a) and (b) is provided to an individual having sufficient fecal threonine fecal levels in a known diet. In some embodiments, the individual is monitored for fecal threonine.
In any of the above embodiments, the autoimmune disease may be inflammatory bowel disease or celiac disease. Inflammatory Bowel Disease (IBD) can be Ulcerative Colitis (UC) and Crohn's Disease (CD). The subject may have hyper inflammatory bowel disease (hyperinflammatory gut). The allergy may be food allergy or atopy.
In any of the preceding embodiments, the subject may be a mammal. The mammal may be a human, cow, pig, rabbit, goat, sheep, cat, dog, horse, llama or camel. The mammal may be an infant. The mammal may be a mammalian infant mammal. The object may be a person.
In any of the above embodiments, the vitamin a can be retinol, retinal, retinoic acid, provitamin a carotenoids, or combinations thereof. The provitamin a carotenoid may be alpha-carotene, beta-carotene, gamma-carotene, lutein beta-cryptoxanthin, or combinations thereof. The microbial a-procarotene may be beta-carotene.
In any of the above embodiments, the Oligosaccharide (OS) comprises at least about 15%, at least 25%, at least 50%, at least 75%, at least 95% of the total dietary fiber of the subject.
In any of the above embodiments, elevating regulatory T cells (tregs) results in the suppression of toxic T helper (Th) cells. Elevation of regulatory T cells (tregs) leads to a decrease in inflammatory markers. The inflammatory marker may be IL-8, IL-6, TNF- α, IL-10INF γ, INF α or a combination thereof. The inflammatory marker is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.
In any of the above embodiments, the subject has been colonized with a bifidobacterium species as measured by bifidobacterium species CFU/gram stool or CFU/μ gDNA. Colonization of bifidobacteria species in the subject may increase stool by at least 1-10 CFU/gram. The subject may not be colonized with a bifidobacterium species as measured by bifidobacterium species CFU/gram stool.
In any of the above embodiments, the dose of retinoic acid or a source thereof, Oligosaccharide (OS), bifidobacterium, or a combination thereof may be an amount effective to maintain a total bifidobacterium level of at least 106At least 108At least 109Or at least 1010Normalized CFU/g feces or μ g DNA, or more preferably greater than 108. Alternatively, the relative abundance of the bifidobacterium family in the microbiome is at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the total measurable microbiome. In some embodiments, the bifidobacterium is bifidobacterium infantis and the effective amount remains greater than 106、107、108、109Or 1010Normalized CFU/. mu.g DNA or gram feces, more preferably greater than 108And (4) CFU. In other alternatives, the metagenome is measured by shotgun sequencing and has an increased abundance of genes compared to individuals not receiving the compositions described herein, including but not limited to Blon2175, Blon2176, and/or Blon 2177.
In any of the above embodiments, the bifidobacterium may be administered to the subject daily, including 10 to 5000 million CFU bacteria per day. The bifidobacteria may be administered to the subject daily, and may comprise from 10 to 1000 or from 50 to 200 million CFU/day. The bifidobacterium may be administered to the subject daily for at least 1,2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days to 365 days. The bifidobacterium may be administered to the subject daily for at least 1-5 days, 6-10 days, 11-15 days, 16-20 days, 21-25 days, 26-30 days, 100 days, or for at least 3 months, 3-6 months, more than 6 months, and more than 1 year.
In any of the above embodiments, the oligosaccharide may be administered in solid or liquid form. Oligosaccharides may be administered in amounts of about 0.1-50 g/day. Oligosaccharides may be administered in amounts of about 2-30 g/day or 3-10 g/day.
In any of the above embodiments, the first composition comprising retinoic acid and oligosaccharide may be administered to a subject. The first composition may be administered multiple times a day, optionally 1-6 times a day. The first composition may be administered for at least 1-365 days.
In any of the above embodiments, the second composition comprising bifidobacterium may be administered to the subject. The second composition may be administered daily. The second composition may be administered for at least 1-365 days.
In any of the above embodiments, the first composition comprising retinoic acid or a source thereof and oligosaccharides may be administered to the subject followed by administration of the second composition comprising bifidobacteria.
In any of the above embodiments, a third composition comprising retinoic acid, oligosaccharides and bifidobacteria may be administered to the subject.
In any of the above embodiments, the vitamin a or vitamin a derivative or metabolite or source thereof may be administered multiple times a day for at least 1-30 days.
In any of the above embodiments, the oligosaccharide may be administered multiple times a day for at least 1-30 days. In other embodiments, the oligosaccharide may be administered for at least 30 days, at least 60 days, at least 90 days, at least 180 days, at least 1 year, or as part of a healthy diet as desired.
In any of the above embodiments, the bifidobacterium may be administered multiple times a day for at least 1-30 days. The vitamin a or vitamin a derivative or metabolite or source thereof, the oligosaccharide and the bifidobacteria may be administered to the subject daily in a composition for at least 1-30 days. Administering vitamin A or a vitamin A derivative or metabolite or source thereof and an oligosaccharide to a subject multiple times daily for at least 1-30 days, followed by administration of bifidobacteria daily for at least 1-30 days.
In any of the above embodiments, the function of the immune system is enhanced in the mammal after administration of the bacterium, the MMO, or both. Enhancing immune system function may improve: vaccine response, mucosal innate or adaptive immunity and/or improving homeostasis of innate and adaptive immunity. In some embodiments, fecal calprotectin is evaluated and elevated levels are indicative of malnutrition.
In any of the above embodiments, immune system function is manifested by: altered B or T cell populations, more specifically, increased regulatory T cells and regulatory B cell populations, increased antibody titers in response to the vaccine, improved mucus production or decreased mucin degradation, or increased secretory immunoglobulin a (siga) production in the gut to confer protection against pathogenic bacteria. The increase may be statistically significant. The increase may be about 5%, 10%, 20%, 30%, 40, 50, 60, 70, 80, or 90%, more preferably 5-20%, 20-40% as compared to a baseline sample of the subject or as compared to an expected value for a subject not receiving a composition described herein.
In any of the above embodiments, the composition can be used to develop and/or enhance an intestinal barrier wherein the proinflammatory cytokines (i.e., TNF α, IL-1 β, and IFN γ) are decreased, ZO-1 and occlusion protein (occlusion protein) are increased, or myeloperoxidase is decreased.
In one embodiment, regulatory T cells (T) are elevatedreg) The compositions of (a) comprise a composition described herein. In another embodiment, the composition alters a helper T cell population, including but not limited to Th 17. In some embodiments, TReg cells are measured and increased. In other embodiments, changes are measured in other T cell populations, including, but not limited to, Th1, Th2, Th17, Th9, or other T cell populations. In some embodiments, the ratio of Treg/Th17 is increased. In other embodiments, Th17 is decreased or TReg is increased.
In some embodiments, with the use of any of the compositions of the present invention, the levels of fecal interleukin 17A (IL-17), IL-8, IL-22, IL-1 β, IL-6, IL-22, TNF α, IL-1 β, and IFN will decrease, or the levels of IL-17, IL-8, IL-22, IL-1 β, IL-6, IL-22, TNF α, IL-1 β, and IFN γ will increase, with dysbiosis. In some embodiments, a value greater than 180pg/mg, greater than 100pg/mg IL-17 is indicative of dysbiosis. In some embodiments, an IL-4 concentration greater than 15pg/mg is indicative of a dysbiosis. In some embodiments, an IL-13 concentration greater than 400pg/mg is indicative of a dysbiosis.
In some embodiments, the adaptive immune system can be measured by assessing B cells and Breg cells, sIgA production and/or vaccine response by antibody titer (including mucosal IgA as well as systemic IgG1 and IgE).
In one embodiment, the method for preventing or treating a symptom of an autoimmune disease, wherein the autoimmune disease may be selected from the group consisting of: inflammatory bowel disease (IBD: including crohn's disease, ulcerative colitis, inflammatory bowel syndrome), Necrotizing Enterocolitis (NEC), atopy, allergy, asthma, celiac disease, autism, type I diabetes, including any of the compositions described herein.
In one embodiment, the subject is a pregnant woman. In some embodiments, the pregnant woman is in her third or fourth trimester.
In embodiments for the prevention or treatment of a metabolic disease, wherein the disease or condition may be selected from: obesity, type II diabetes or processes involving cognitive development (learning, depression).
In one embodiment, the composition for supporting (adjuvanting) cancer treatment comprises a composition as described herein.
In one embodiment, a composition for use in maintaining gut mucosal integrity during chemotherapy or extreme chemical reduction of microbiome in the case of recurrent Clostridium difficile (c.difficile) infection, the composition comprising a composition described herein. In some embodiments, a relapsing clostridium difficile or other partial infection (reactive infection) is treated with a composition described herein before or after fecal transplantation.
In some embodiments, the target population is human infants with dysbiosis gut microbiome. In other embodiments, the compositions improve the efficacy of vaccine responses and immune system targeted therapies intended to improve the health of individuals of any age.
All the applications of the present invention can be used for preventing and/or ameliorating an inappropriate response to a condition caused by: pregnancy, childbirth, preterm labor, colic, diaper rash, sleep, weaning to eating aids, weaning from breast milk or from formula milk to solids, mucosal damage, atopic diseases, food allergies, autoimmune diseases, metabolic disorders, cognitive development, obesity, pre-and post-fecal transplantation therapy, gene therapy, immunotherapy or vaccine reactions.
Brief description of the drawings
FIG. 1. PCoA of upper intestinal microbiome at family level; control (CON) samples are shown as gray triangles, while EVC001 fed infant samples are shown as light gray circles. The overall variation of 87.5% was described in the first two principal components (PC1 and PC 2). The PERMANOVA comparison identified significant differences in composition between the two treatment groups (R. RTM. 26.5, P. RTM. 0.001).
FIG. 2 comparison of fecal carbohydrate groups and colonic mucin-derived O-glycans of feces from control and EVC001 fed infants. (A) Total number of OS detected between treatment groups. (B) The number of colonic mucin-derived O-glycans between treatment groups. (C) Relative abundance of total number of colonic mucin-derived O-glycans in total OS pools (pool) between treatment groups. (D) Of the total OS abundance between treatment groups, the OS was assigned as a percentage of colonic mucin-derived O-glycans.
FIG. 3 PCoA of colonic mucin-derived O-glycan compositions between samples using a Bray-Curtis dissimilarity index; control (Con) samples are shown as grey triangles, whereas EVC001 fed infant samples are shown as light grey circles, with a total variation of 63.3% described in the first two principal components (PC1 and PC 2). For colonic mucin-derived O-glycan compositions, the PERMANOVA comparison identified significant differences that existed between the two treatment groups (R ═ 12.4, P ═ 0.001).
Figure 4. relative abundance of specific gut groups after birth. The box plots represent the top 10 most abundant intestinal clusters (gut taxa) in control and EVC001 fed infants on (a) day 6, (b) day 40, and (c) day 60. If P is less than 0.05; p < 0.01; p < 0.001; p < 0.0001, P values were considered statistically significant.
Fig. 5A-b stool calprotectin levels depend on the abundance of bifidobacterium family (bifidobacterium). Fecal calprotectin concentrations and bifidobacteraceae abundances (a) were evaluated in 40 fecal samples at postnatal day 40 and subdivided according to bifidobacteraceae abundances < or > 25% (B). The data set represents at least three different experiments performed in triplicate and significance is determined using the nonparametric Vickers (Wilcoxon) rank-sum test, with corresponding P values adjusted and considered statistically significant if Pop < 0.0001.
Figure 6 faecal cytokine profile of infants receiving bifidobacterium infantis EVC 001. Radar plots represent median cytokine concentrations [ pg/mg ] tested on fecal samples from post-natal (a) day 6 (baseline), (b) day 40 post-natal, and (c) infant fed bifidobacterium infantis EVC001 at day 60 (EVC001) or control (n ═ 20). The median was adjusted to a logarithmic scale and then normalized from 0-1 in each cytokine group. Statistical analysis was done using verxon rank sum test. Adjusting the P value using the Bonferonni-Holm method, and if P < 0.05; p < 0.01, considered statistically significant.
FIG. 7: fecal cytokine concentrations change after birth. The box plots represent fecal proinflammatory cytokine concentrations [ pg/mg ] for (a) IL-2, (b) IL-5, (c) IL-6, (d) IL-8, (e) IL-10, (f) IL-22, (g) TNF α, (h) IL1 β, and (i) IFN γ from day 6 (baseline), day 40, and day 60 control (n-20), and EVC001 fed infants (n-20). Cytokine concentrations were measured in duplicate using a MesoScale discovery U-plex. Statistical analysis was done using verxon rank sum test. Adjusting the P value by using a Bonferonni-Holm method, if P is less than 0.05; p < 0.01; p < 0.001; p < 0.0001, P values were considered statistically significant.
FIG. 8: principal coordinate analysis (PCoA) of global cytokine profiles from group status. PCoA is based on Bray-Curtis similarity of overall cytokine profiles between post-natal (a) day 6 (baseline), (B) day 40, (C) EVC001 fed infants and controls at day 60.
FIG. 9: correlation between specific gut groups and gut inflammatory cytokine responses. The heat map shows the correlation between the family of bacteria and specific cytokines calculated by Spearman correlation P-values corrected using the Benjamini-Hochberg program (FDR) to estimate significant correlations between specific cytokine concentrations and microbiologic composition detected in the faeces of breast-fed infants only at three times (day 6 (baseline), day 40 and day 60 after birth). Each cytokine was tested in duplicate at three different time points. Adjusting the P value and if P < 0.05 (open circles); p < 0.01 (semi-solid circle); tp < 0.001 (filled circle), the P value is considered statistically significant.
Figure 10 relationship between sIgA production and relative abundance of bifidobacterium family.
FIG. 11 is a block diagram representing the concentration of the fecal proinflammatory cytokines IL-17A, IL-13 and IL4 [ pg/mg ] from controls at day 60 post-natal. Cytokine concentrations were measured in duplicate using a MesoScale discovery U-plex.
FIGS. 12A-D depict variations in the following: (A) spleen weight; (B) cecal weight; (C) lymphocyte counts in the spleen; (D) lymphocyte counts in Mesenteric Lymph Nodes (MLN) from humanized mice treated with LNnT alone or in combination with bifidobacterium infantis.
FIGS. 13A-F depict analysis of lymphocyte populations in untreated and treated mice; (A) total lymphocytes; (B) CD4 +; (C) CD4+/CD25+ Helios-FoxP3 +; (D) CD4+/CD25+/Helios-FoxP 3-; (E) CD4+/FoxP3+ CD 25-; (F) CD4+/Helios + CD 25-.
FIG. 14 depicts colony forming units of Bifidobacterium infantis and Enterobacter in mice after 21 days.
FIG. 15 depicts the treatment groups for the SAM assay of example 12.
FIG. 16 depicts the stratification of subjects in the study described in example 16.
Detailed description of the invention
As described in international application PCT/US2018/050973, creating a healthy intestinal environment is critical to the overall health of a mammal. The inventors have discovered a means of providing or removing key metabolites and/or precursors thereof from the intestine in an amount sufficient to alter the entire intestinal metabolome. The abundance of key metabolites may play a role in nutrition, absorption, metabolism, and immune function to promote the overall health of the mammal. These metabolites may also be given therapeutic capacity to restore homeostasis in the event of alterations in metabolism (i.e. obesity, type 2 diabetes, necrotizing enterocolitis), cognitive function (i.e. cognitive development, learning, depression, autism), autoimmunity (i.e. celiac disease, type I diabetes, atopy, allergy) or inflammation (i.e. inflammatory bowel disease, irritable bowel syndrome).
These metabolites can be increased or decreased, alone or in combination, to modulate physiology, immunity and biochemistry of the infant gut, as described in more detail in international application PCT/US 2018/050973. The above application describes compositions, methods and regimens that provide sufficient levels of these compounds to restore and promote the nutritional and metabolic health of the intestine and the health of other critical organs, including the liver and central nervous system. Monitoring the status of some or all of the metabolites may be used to identify people who will be at risk of developing disease in the future.
Typically, the key ingredient is delivered by administering to an animal, more particularly to a mammal, even more particularly to a human, a composition comprising retinoic acid or a source thereof and an Oligosaccharide (OS) which is a Mammalian Milk Oligosaccharide (MMO) or a functional equivalent thereof. These compositions may be administered in combination with a bacterial composition comprising a bacterium that expresses a key exopolysaccharide on its cell surface, and may be activated to utilize the OS in the composition.
Preparation and application of vitamin A or vitamin A derivative
In some embodiments, dietary vitamin a is delivered as pre-formed (preformed) vitamin a or pro-vitamin a as part of the diet. In other embodiments, vitamin a is supplemented outside of its typical diet to increase vitamin a consumption. The vitamin a that has been formed is described as being from meat, poultry, fish or dairy products, whereas vitamin a was originally derived from plant sources. Vitamin a deficiency is rare in the united states; however, it may be a problem in premature infants and in less developed countries. In some embodiments of the invention, the composition will comprise about 2.3 μmol/l vitamin A. In other compositions will comprise at least 1 μmol/l vitamin a, and in other compositions the vitamin a may range between 0.4-1.2 μmol/l). In other embodiments, the subject has a target vitamin A concentration of 6-10 μmol/l. Vitamin a (retinol) is ingested in the form of retinyl esters or carotenoids and is metabolized into active compounds such as 11-cis-retinal and all-trans-retinoic acid.
Retinoids are typically isolated from animal sources, while carotenoids are typically isolated from plant sources-in some embodiments, the source of vitamin a is provided in the form of retinoic acid or other derivatives, and can be used to stimulate T regulatory cells (tregs). In other embodiments, precursors to retinoic acid from the retinoid compound family are provided. In other embodiments, the plant carotenoid is delivered with the bacterial composition and converted to retinoic acid or retinol by the gut microbiome. In other embodiments, alpha-carotene, beta-carotene, gamma-carotene, and beta-cryptoxanthin and astaxanthin are examples of plant carotenoids provided, and may be converted to retinoic acid under certain conditions in the intestine, whether from host heritability or microbiome. In some embodiments, a source of carotenoids is used. In other embodiments, a source of retinoid is used, and in other embodiments, a combination of a carotenoid and retinoid in a ratio of 1: 10 to 10: 1 provides a method of controlling retinoic acid availability (e.g., time-released) to maintain a constant source of retinoic acid. In some embodiments, the carotenoid is considered a slow-release retinoic acid, while the retinol is considered a fast-release or bioavailable source. In some embodiments, the composition is formulated to release retinoic acid in the small intestine, while in other embodiments, the composition is formulated to release retinoic acid in the large intestine or colon. Vitamin a can be expressed in international units. International units can be converted to mg vitamin a. Vitamin a or provitamin a may also be discussed in terms of Retinol Activity Equivalent (RAE). The present invention provides for the intake of vitamin a: 700 micrograms per day (mcg) of Retinol Activity Equivalent (RAE) for persons 14 years of age and older; 1,200-1,300RAE per day for lactating women; 1500 to 2500IU for infants and children under 3 years of age, and about 6,000 IU for adults over 19 years of age.
One aspect of the present invention calls for increasing the bioavailability of retinol and/or increasing the conversion to retinoic acid, which may not be achievable for a particular age group or gender, with the generally recommended vitamin a levels. In subjects requiring such intervention, the ability to stimulate, tolerate and/or expand the TReg population may require a conditional increase in the bioavailable vitamin a sources, such as vitamin a and/or provitamin a that have been formed, to increase the ability to metabolically convert to retinoic acid, thereby meeting metabolic requirements. In other embodiments, where it is known or expected that an individual will lack vitamin a, preparing the composition will include calculating the requirements of an individual that will consume an amount of vitamin a and/or provitamin that has developed to meet or exceed a threshold in the individual's diet. In other embodiments, the ratio of retinoid to carotenoid is determined so as to provide a sustainable increase in retinoic acid in the individual. In other embodiments, serum levels of retinoic acid are monitored to achieve a constant state.
Compositions and formulations of oligosaccharides
The OS composition (structures present) and its amount (grams) may support the colonization and activation of bifidobacterium infantis. The OS composition may maintain the activation of bifidobacterium infantis.
The term "oligosaccharide" as used herein broadly refers to any oligosaccharide having 3-20 residues, regardless of the source of the oligosaccharide.
lacto-N-disaccharide (LNB) is a moiety that is the core of an oligosaccharide or may be the entity itself. It may be in a type I or type II core configuration, meaning a β 1-3 or β 1-4 linkage, respectively. N-acetyl lactosamide is an example of a type II entity. LNnT is an example of a larger oligosaccharide structure comprising a type II core. An example of a larger type I core is an LNT.
As used herein, "source(s) of oligosaccharides" broadly refers to oligosaccharides in the form of free oligosaccharides derived from animals, insects, crustaceans, microorganisms, plants, fungi or algae or chemically synthesized, as well as those associated with animal or plant proteins or lipids (glycans), and those glycan structures following their release from proteins or lipids or mixtures thereof.
The term "mammalian milk oligosaccharide" or MMO, as used herein, refers broadly to those indigestible glycans, sometimes referred to as "dietary fiber", or carbohydrate polymers that are not hydrolyzed by endogenous mammalian enzymes in the mammalian digestive tract (e.g., small intestine). Mammalian milk contains a significant amount of MMO, which cannot be used directly as an energy source for feeding mammals, but can be used by many microorganisms in the intestine of such mammals. MMOs can be free oligosaccharides (3 saccharide units or longer, e.g. 3-20 saccharide residues), or they can be conjugated to proteins or lipids.
In some embodiments, optionally, the composition comprises a bacterial cell wall exopolysaccharide. In some embodiments, the living cells are used to provide exopolysaccharides. In other embodiments, dead cells are used to provide exopolysaccharides. In other embodiments, a combination of live and dead cells is used.
The OS, which includes MMOs and functional equivalents thereof, such as, but not limited to, isolated white natural milk MMOs, synthetic equivalent natural MMOs, modified plant or fungal polysaccharides, modified animal, insect or crustacean polysaccharides, or glycans released from animal or plant glycoproteins (i.e., milk, meat, eggs, fish, soy, corn, peas), supports the growth and metabolic activity of these bacteria, and thus can be used in the present invention.
Mammalian milk contains a large amount of Mammalian Milk Oligosaccharides (MMO) as dietary fibers. For example, in human milk, dietary fiber is about 15% of the total dry weight, or about 15% of the total caloric content. These oligosaccharides comprise sugar residues in a form that cannot be used directly as an energy source for most microorganisms in a mammalian infant or adult or in the intestine of that mammal.
The MMO may be provided to the mammal in the form of a food composition. The food composition may comprise mammalian milk, a mammalian milk-derived product, mammalian donor milk, infant formula, a milk substitute, or an enteral nutrition product or meal replacement for mammals including humans. In some embodiments, the addition of the food composition comprising MMO and the bacterial composition may occur simultaneously, e.g., within less than 2 hours of each other.
MMOs for use in the present invention may include Fucosyllactose (FL) or derivatives of FL, including, but not limited to, lacto-N-fucopentaose (LNFP) and lacto-difucotetraose (LDFT), which may be neutral, such as, but not limited to, N-acetyllactamide, lacto-N-disaccharide (LNB), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which may be purified from mammalian milk, such as, but not limited to, human milk, cow milk, goat or horse milk, sheep or camel milk, or produced directly by chemical synthesis. The composition may further comprise one or more bacterial strains having the ability to grow and divide using fucosyllactose or derivatives thereof as sole carbon source. Such bacterial strains may be naturally occurring or genetically modified and may be selected to grow on fucosyllactose or derivatives thereof if such bacterial strains do not naturally grow on those oligosaccharides.
The MMO may also be Sialyllactose (SL) or derivatives of SL such as, but not limited to, 3 'sialyllactose (3SL), 6' sialyllactose (6SL) and disialyllacto-N-tetraose (DSLNT), which may be purified from mammalian milk such as, but not limited to, human milk, bovine milk, goat or mare milk, sheep or camel milk, or produced directly by chemical synthesis. The composition further comprises one or more bacterial strains having the ability to grow and divide using sialyllactose or a derivative thereof as the sole carbon source. Such bacterial strains may be naturally occurring or genetically modified and may be selected to grow on sialyllactose or derivatives thereof if such bacterial strains do not naturally grow on those oligosaccharides.
The MMO may be Fucosyllactose (FL) or a mixture of FL and Sialyllactose (SL) or SL derivatives, which are naturally present in mammalian milk, such as, but not limited to, human milk, bovine milk, goat milk, and horse milk. FL and SL or a derivative thereof may be present in a ratio of about 1: 10 to 10: 1.
The selective Oligosaccharides (OS) as defined herein are carbohydrates that are not digested by mammals and are more favorable for the growth of specific bacteria than others. The selective oligosaccharides may be of mammalian milk, plant, algal, yeast origin, as long as they induce the desired metabolic properties. OS, as used herein, refers to those indigestible sugars from any source, including plants, algae, yeast or mammals, having a length of DP2-DP 20. Oligosaccharides having the chemical structure of indigestible oligosaccharides present in any mammalian milk are referred to herein as OS, whether or not they are actually derived from mammalian milk.
OS may include one or more of the following: lacto-N-disaccharide, N-acetyllactosamide, lacto-N-trisaccharide, lacto-N-neotetraose, fucosyllactose, lacto-N-fucopentaose, lactodifucotetraose, sialyllactose, disialolactone-N-tetraose, 2 '-fucosyllactose, 3' -sialyllactosamine, 3 '-fucosyllactose, 3' -sialyllactose, 6 '-sialyllactosamine, 6' -sialyllactose, difucosyllactose, lacto-N-fucosylpentaose I, lacto-N-fucosylpentaose II, lacto-N-fucosylpentaose III, lacto-N-fucosylpentaose V, sialyllacto-N-tetraose, or a derivative thereof. In some embodiments, the OS includes a type I core. In a preferred embodiment of the mixture, the OS comprises a type II core. See, for example, U.S. patent nos. 8,197,872, 8,425,930, and 9,200,091, the disclosures of which are incorporated herein by reference in their entirety. Functional equivalents of MMOs can include the same molecules produced using recombinant DNA techniques such as described in australian patent application publication No. 2012/257395, australian patent application publication No. 2012/232727 and international patent publication No. WO 2017/046711.
In general, plant and fungal fibers are such polysaccharide structures that can only be digested extracellularly by colonic bacteria that excrete certain hydrolases, and then digest the free sugar monomers or oligosaccharides produced by extracellular hydrolysis. However, enzymatic, chemical or biological treatment of plant and fungal fibers can reduce the size of glycans to a size that can be utilized by certain bacteria capable of digesting and deconstructing MMO, such as but not limited to Bifidobacterium longum (B.longum and B.breve), furthermore, the present invention contemplates treatment with synthetically and/or recombinantly produced hydrolases that mimic microbial carbohydrate hydrolases, such as GH5, GH13, GH92, GH 29.
Chemical treatments of plant polysaccharides include acid hydrolysis (sulfuric acid, hydrochloric acid, uric acid, trifluoroacetic acid), or hydrolysis using acidic hydrophobic, non-aqueous, ionic liquids, followed by separation of the oligosaccharides in a two-phase reaction using water (Kuroda et al, ACS supersteable chem. eng., 2016, 4(6), p. 3352-3356). Polysaccharides or glycans attached to proteins or lipids can be released by enzymatic processes using N-linked and/or O-linked glycans.
In some embodiments, chitin or chitosan may be derived from crustacean or fungal sources (i.e., shrimp and shiitake), and may be processed to deliver structures of DP 2-20 for use in certain compositions.
The formulation may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% N-acetyl-D-lactosamine (dimer; type II core, typically in LNnT). For example, a formulation may comprise about 5% -95%, 10% -80%, 50% -75% or 20% -60% N-acetyl-D-lactosamine (dimer; type II core, typically in LNnT). In addition, the formulation may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of a core type I HMO (Gal- (1, 3) - β -GlcNAc) synthesized by an enzyme having homology to β -3-galactosyltransferase 1(B3GALT1) present in the human genome. For example, a formulation may comprise about 5% -95%, 10% -80%, 50% -75%, or 20% -60% core HMOs type I (Gal- (1, 3) - β -GlcNAc). Oligosaccharides not present in human milk, such as dimeric structures found during synthetic production of oligosaccharides or other intermediate dimers, including disaccharides, e.g., lacto-N-disaccharide, may be used. The formulation may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% lacto-N-trisaccharide I (Gal- (1, 3) - β -GlcNAc- (1, 3) -Gal) or lacto-N-trisaccharide II (GlcNAc- (1.3) -Gal- (1, 3) - β -Glu) or lacto-N-neotrisaccharide (Gal- (1.4) - β -GlcNAc- (1, 3) -Gal). For example, a formulation may comprise about 5% -95%, 10% -80%, 50% -75% or 20% -60% lacto-N-trisaccharide I (Gal- (1, 3) - β -GlcNAc- (1, 3) -Gal) or lacto-N-trisaccharide II (GlcNAc- (1, 3) -Gal- (1, 3) - β -Glu) or lacto-N-neotrisaccharide (Gal- (1, 4) - β -GlcNAc- (1, 3) -Gal). The MMO may provide 0.2 grams to 40 grams per day.
MMOs or similar selective oligosaccharides used at a percentage of greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% are diluted to a percentage of less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 0.40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% in non-selective oligosaccharides such as, but not limited to, galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), xylo-oligosaccharides (XOS), or combinations thereof. These combinations provide a degree of increased selectivity, where the higher the proportion of MMO or source of selective oligosaccharide structures, the greater the selectivity towards certain bacteria, such as but not limited to bifidobacterium longum subsp.
Modification of the oligosaccharide structure to increase sialylation (sialyllactosamine) or fucosylation may further improve their selectivity. The formulation may comprise a type II core dimer of lactosamine, and fucosylated and/or sialylated oligosaccharides as the selective oligosaccharide moiety, with the remainder consisting of non-selective or low-selective oligosaccharides. The composition may be formulated to further include vitamin a, a vitamin a derivative or metabolite, the amount of which is adjusted relative to the OS content of the composition.
The term "synthetic" composition refers to a composition that is produced by a chemical synthetic process and may be identical in nature. For example, the composition may include chemically synthesized and purified or isolated components. This does not include naturally synthesized compositions.
Purification of oligosaccharides may mean separation of one component of milk from any other component or other processing of mammalian milk including expression of human milk to provide, for example, partially skimmed pre-milk, human donor milk or other human milk products such as fortifiers (fortifiers).
The OS may be provided to the mammal directly or in the form of a food composition. The composition may further comprise a food, and the food may comprise part or all of the nutritional requirements to support the life of a healthy mammal, wherein the mammal may be, but is not limited to, an infant or an adult. The food composition may comprise mammalian milk, a mammalian milk-derived product, mammalian donor milk, infant formula, a milk substitute, an enteral nutrition product or a meal replacement for mammals including humans. OS may be in the form of a powder or liquid (water-based or oil-based), gel, or paste.
Compositions and formulations of bifidobacteria
In any of the preceding embodiments, the composition may further comprise bifidobacteria. The Bifidobacterium may be Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium animalis subsp.animalis, Bifidobacterium animalis subsp.lactis, Bifidobacterium bifidum (b.bifidobacterium), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium catenulatum (Bifidobacterium catenulatum), Bifidobacterium longum subsp.infantis, Bifidobacterium pseudocatenulatum (b.pseudocatenulatum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), or a combination thereof. The composition may comprise activated bifidobacteria. The bifidobacterium longum may be bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis may be an activated bifidobacterium longum subspecies infantis. Extracellular polysaccharides and solute-binding proteins on the cell surface of bifidobacterium infantis may be increased. The bifidobacterium may be bifidobacterium breve. The bifidobacterium breve may be activated bifidobacterium breve.
In any of the embodiments above, the bacterium can be bifidobacterium longum subspecies infantis EVC001 deposited under ATCC accession No. PTA-125180; cells were deposited from the Budapest treaty on microbial preservation internationally recognized for patent procedures, at the American Type Culture Collection ("deposited bacteria") in the University of Marnsas 10801, Va.virginia, 10801 University Blvd, Manassas, VA 20110, USA.
Furthermore, as used herein, "deposited bacterium" refers to an isolated Bifidobacterium longum subsp. longum infant subsp. infanis EVC001 (deposited with the ATCC and assigned an access number) and variants thereof, wherein said variants retain the phenotypic and genotypic characteristics of said bacterium, and wherein said bacterium and variants thereof have LNT transport capability and comprise a functional H5 gene cluster comprising at least BLON2175, BLON2176 and BLON 2177.
Functional "H5 cluster" refers to the cluster of genes responsible for human milk oligosaccharide uptake and metabolism in bifidobacteria. The functionality H5 cluster contains Blon _2175, Blon _2176, and Blon _ 2177. The H5 cluster contains the following genes: blon _2171, Blon _2173, Blon _2174, Blon _2175, Blon _2176, Blon _2177, and galT.
Activation/activation is defined as a means to switch on a specific nutrient consuming phenotype in bacteria (such as the HMO phenotype in bifidobacterium infantis) during bacterial production, where the bacteria are dry in this state, examples of which include in international patent application PCT/US2015/057226 filed 2015, 10, 23 and international application PCT/US2019/014097 filed 2019, 1, 18.
Activated bifidobacterium infantis appearanceState of the surface
The bacteria may be administered simultaneously with the OS, or they may already be present in the intestinal tract of the mammal. Unlike most intestinal flora, certain important bifidobacteria, such as but not limited to bifidobacterium longum subsp. The functional range may preferably be further defined as 2-10 sugar moieties. This feature has led to the unique success of these bifidobacteria in colonising the gut of breast-fed infants, with oligosaccharides (denoted MMO herein) of appropriate size and correct composition for consumption by these bacteria alone. Such structures are also found in certain plant and animal glycoprotein carbohydrate components. The inventors have also found that these glycans can also serve as mimetics of MMO when they are released from their corresponding glycoproteins. Such oligosaccharides are preferentially internalized and metabolized by such bacteria due to their unique genetic capabilities. Oligosaccharides may be present in mammalian milk but may also be of synthetic or plant origin, as long as they have the ability to select for a particular organism that can provide the nutritional ingredients required for growth and/or development of an infant mammal.
Specific exopolysaccharide gene clusters are found in bifidobacterium longum, which produce characteristic branched exopolysaccharides with unusual partial deoxy-L-talose that appear in a dense coating that completely covers the organism (Altmann et al PLOS one (2016)11 (9): e 0162983). This same gene cluster is not present in bifidobacterium infantis-six essential genes forming this structure are not present in bifidobacterium infantis ATCC 15697 (table 1). Table 2 describes BLAST comparisons of selected proteins between two bifidobacterium species as a strategy to identify cell surface components characteristic of bifidobacterium infantis. In table 2: cadent is the percent identity between two amino acid sequences calculated by BLAST, length is the length of the alignment of the amino acids, mismatch is the number of mismatches between the two sequences, gap is the number of gaps BLAST nicks to cut for optimal alignment, qstart is the start of the alignment in the query sequence, qnend is the end of the alignment in the query sequence, and Sstart is the start of the alignment in the subject sequence. In some embodiments, the bifidobacterium infantis-specific exopolysaccharide is expressed. In some embodiments, the cell membrane of bifidobacterium infantis is used in the composition of the invention.
Table 1: deoxy-L-talose gene cluster
Figure BDA0002927731740000231
Figure BDA0002927731740000241
Figure BDA0002927731740000251
In some embodiments, the composition comprises a high abundance of bifidobacterium infantis of solute binding protein family 1(F1 SBP). The inventors have found that when bifidobacterium infantis is present in a composition that expresses certain unique exopolysaccharides or solute-binding proteins, or comprises key bacterial membrane components, the composition comprising Oligosaccharides (OS) and retinoic acid fed to an individual will act synergistically to develop the immune system or reset an aberrant immune response, in particular promoting its expansion and/or tolerance by T regulatory (TReg) cells.
In some embodiments, a layer of exopolysaccharides specific for bifidobacterium infantis and a membrane fraction or lysed cell membranes from intact dead cells may be included as part of the composition to increase immune stimulation.
Composition comprising threonine
In some embodiments, the amino acid threonine or N-acetyl threonine and/or the peptide gamma-glutamyl threonine are included in the composition at higher levels and are measured in the feces of individuals consuming the composition, and mucin production levels can be greater than mucin degradation levels. In other embodiments, administration of the composition results in a thicker mucus layer on the cell surface. In some embodiments, the state of mucin degradation in the stool is monitored by looking for the amount of certain mucin structures in the stool, or by the presence or absence of certain mucin-degrading bacteria.
Method for altering the immune system
Within the T cell population, altering the regulatory T (treg) cell population is an important component of the present invention. A stronger intestinal barrier and/or appropriate B-cell and T-cell populations may increase the efficacy of a given treatment on an infection or disease and/or may improve colonization of the gut microbiome and/or may reset and/or improve tolerance to food antigens. In addition to changes in TReg cells, suitable T cell populations may include changes to Th1, Th2, Th17, Th9, or other T cell populations. Tregs also have the ability to inhibit B cell and plasma cell responses, resulting in inhibition of B cell mediated disease progression, particularly autoimmunity. Tregs play an important role in controlling the immune response of B and T cells that are specific for the autoantigen that causes autoimmunity. In addition, B regulatory cells (bregs) have the ability to inhibit CD4+ T cells.
IL-17A is one of six different cytokines, included in the cytokine family IL-17, and is often referred to as IL-17 only. It is predominantly expressed by different types of T cells, T helper 17(Th17) cells, and can be less expressed by other specific lymphocytes, including Th17, NK T cells, macrophages and Paneth cells, to mediate pro-inflammatory responses and provide protection at epithelial and mucosal sites in host defense. Although IL-17 production is critical for acute inflammation and to protect the host from pathogen attack, long-term production of IL-17 may lead to excessive expression of proinflammatory cytokines and chronic inflammation, leading to tissue damage and autoimmunity. IL-17 cytokines have been associated with a number of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis. In some cases, IL-4 or IL-13, IL-8, IL-22, IL-1 β, IL-6, IL-22, TNF α, IL-1 β, and IFN γ can be measured.
IL-17A can cause and/or exacerbate a fetal inflammatory response that increases neonatal morbidity and mortality from common neonatal diseases, including sepsis, bronchopulmonary dysplasia, patent ductus arteriosus, and necrotizing enterocolitis. In some embodiments, reducing IL-17A production can reduce neonatal morbidity and mortality.
The present invention includes, but is not limited to, increasing the bioavailability of threonine, N-acetyl threonine or gamma-glutamyl threonine, as further described in U.S. provisional patent application No. 62/558,349, to promote mucus production and reduce mucin-degrading microbiome material. In some embodiments, one or more components are used as part of a treatment regimen, and their composition may vary over time.
The methods described herein can increase the function of the immune system in a mammal, such as improving vaccine response, tolerance to microbial and food antigens, and/or mucosal innate or adaptive immunity, and/or improving the production and transfer of secretory IgA in the gut of a mammal. The enhancement of immune system function is shown, for example, by: in response to vaccine antibody titer enhancement, mucus production is improved, T-and B-regulatory cell populations are increased, or sIgA production is increased to confer protection against pathogenic bacteria. The increase in immune system response may be statistically significant. For example, the reaction may be increased by 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
Immune function may be selected for their ability to alter receptors such as pattern recognition receptors (i.e. Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4), NOD-like receptor Farnesoid X Receptor (FXR), TGR5 or aryl hydrocarbon receptor (AhR)).
The immunological modification may comprise a COX-2 reduction.
Age related status
Enhancement of the immune system through B cell development (including, but not limited to, B cell maturation and plasma cell development) is important during pregnancy, preterm birth, infants, colics, diaper rash, weaning, immunotherapy treatment and vaccine response in infants and adults (over 55 years of age).
Disease states
Many different autoimmune disorders, inflammatory disorders and therapies requiring a functional immune system can be improved by using the compositions described herein, including, but not limited to, Inflammatory Bowel Disease (IBD), including crohn's disease and ulcerative colitis, and Inflammatory Bowel Syndrome (IBS), Necrotizing Enterocolitis Colitis (NEC), allergies, atopy, obesity, type 1 diabetes, type II diabetes, vaccine reactivity, autism, organ transplantation, immunotherapy and gene therapy.
IBD is a generic term describing diseases involving chronic inflammation of the intestine. Treatment of IBD often requires anti-inflammatory or immunosuppressive drugs (with significant side effects) and/or surgery (with lifetime morbidity). For example, Ulcerative Colitis (UC) is a condition that causes long-term inflammation and ulcers (ulcers) in the innermost layers of the large intestine (colon) and rectum, and over 50% of patients require surgical resection of the entire colon and rectum. There is a need for alternative safer interventions to suppress overly inflamed intestinal mucosa. Atopy and allergy are terms that include many conditions resulting from hypersensitivity of the immune system to environmental allergens. These allergens are usually proteins that have little problem for most people. Treatment of allergy includes treatment of mild allergy with antihistamines, and intramuscular injection of epinephrine to those with more severe complications. For example, Anaphylaxis (Anaphylaxis) is a severe allergic reaction that occurs rapidly and can lead to death. New treatments, such as oral immunotherapy, which introduce low levels of allergens to children known or expected to have peanut allergies, have been shown to be expected to reintroduce peanut allergens by diet. The present invention provides compositions useful for preventing or treating allergy, which prevent or slow down the hypersensitivity immune system and induce tolerance.
Atopic progression (Atopic March) refers to the typical development and progression of allergic disease in early life. These include atopic dermatitis (eczema), food allergy, atopic wheezing, asthma and allergic rhinitis. It is also commonly referred to as the Allergic history (Allergic March).
Type I diabetes, referred to as type 1 diabetes, is a chronic metabolic disease in which high levels of glucose are found in the blood, leading to poor health outcomes. The present invention provides compositions that can be used prophylactically to induce tolerance, thereby avoiding autoimmune reactions that result in the destruction of insulin-producing cells in the pancreas.
Gene therapy and vaccine responses are not usually tested in view of their past strong efficacy; however, it is now clear that the gut microbiome composition has an impact on the vaccine response of infants and young children, which makes them more susceptible to illness and death. The present invention provides compositions that can be used for alternative safer interventions to improve vaccine efficacy.
Supplementation and treatment regimens
The compositions of the invention may be administered for at least 24 hours, at least 72 hours, at least 21 days, at least 28 days, at least 12 weeks, 16 weeks, 6 months or at least 1 year to develop robust and appropriate immune modifications. The treatment is designed to stimulate the immune system for the purpose of improving host defenses, including, but not limited to, improving mucus production and/or reducing mucus degradation, B cell reactivity and/or augmenting or altering T regulatory and helper T cell profiles. The composition can result in the induction of oral tolerance and improved vaccine efficacy.
The composition can be a food composition sufficient to provide part or all of the nutritional source to a mammal, and can include a threonine-rich protein source. The bacteria and the oligosaccharide, e.g., an increased metabolite, such as threonine, N-acetyl threonine, or gamma-glutamyl threonine, and a decreased metabolite, such as retinol (vitamin a), are administered separately or in a food composition in amounts sufficient to maintain the desired level and composition of at least one metabolite in the mammal. A complete list of metabolites can be found in U.S. provisional patent application serial No. 62/558,349. Other examples of altered metabolites are found in international patent application No. PCT/US2017/040530 filed on 30.6.2017 and US provisional patent application No. 62/613,405 filed on 3.1.2017.
The following examples are provided to illustrate various ways of the invention disclosed herein, but they are not intended to limit the invention in any way.
Examples
Example 1
Feeding Bifidobacterium infantis EVC001 to infants consuming a HMO-enriched diet
The test was intended to show the effect of supplementation of probiotic bacteria with bifidobacterium longum subspecies infantis (bifidobacterium infantis EVC001) compared to the unsupplemented group in healthy term care infants. Dry compositions of lactose and activated bifidobacterium longum subspecies infantis were prepared starting from purified isolates (strain EVC001, ATCC accession No. PTA-125180, evolved Biosystems Inc (evolvent Biosystems Inc.), davis, california, isolated from human infant stool samples) cultured in the presence of BMO according to international patent application No. PCT/US 2015/057226. The cultures were harvested by centrifugation, lyophilized, and the concentrated powder preparation had about 3000 billion CFU/g of activity. The concentrated powder was then diluted to an activity level of about 300 billion CFU/g by mixing with infant formula grade lactose. The composition is then filled into individual pouches at about 0.625 g/pouch and provided to breastfed infants starting on day 7 or about 7 of birth, daily for the next 21 days.
This is a 60-day study, starting on the birth date of the infant, i.e. day 1. Women and their infants (either delivered by syn or cesarean section) were randomized to unsupplemented or bifidobacterium infantis + breastfeeding support before postnatal day 6. There were no differences in infant birth weight, birth length, gestational age at birth and gender between supplemented and unsupplemented groups. Infants in the supplementary group were given at least 1.8x10 suspended in 5mL of breast milk once a day starting on postnatal day 7 and continuing for 21 days thereafter10Dose of cfu bifidobacterium infantis. Due to HMO supplied by breast milkSupport for colonization by bifidobacterium infantis is of crucial importance, so all participants receive breast-feeding support in hospitals and homes and remain purely breast-fed during the first 60 days of life. The complete study design is described in [ Smilowitz et al BMC Pediatrics (2017) 17: 133DOI 10.1186/s12887-017-0886-9]。
Throughout the 60 day trial, samples of infant faeces were collected. Mothers collected their own stool and breast milk samples as well as stool samples from their babies. They filled out a health and diet questionnaire once a week, two weeks and a month, as well as daily logs on their infant feeding and gastrointestinal tolerance (GI). Safety and tolerability were determined by mother's reports on infant feeding, frequency of bowel movements and consistency (using the modified Amsterdam infant stool scale) -waterborne (water), soft, formed (formed), hard; Bekkali et al 2009) and GI symptoms and health outcomes. Complete microbiome analysis was performed on each stool sample using 16S rDNA-based Illumina sequencing and qPCR with primers designed for bifidobacterium longum subspecies infantis.
Bifidobacterium infantis was determined to be well tolerated. The reported adverse events were those expected in normal healthy term infants and were not different between groups. Reports specifically monitor blood in the infant's stool, infant body temperature, and parental assessments of GI related infants, such as general irritability, discomfort from vomiting and passage of stool or gas, and flatulence. In addition, no difference was seen in the reported parental reports of medical diagnosis of antibiotic use, carminative medication, or infant colic, jaundice, number of diseases, illness visits, and eczema.
Regardless of the mode of delivery (either antenatal or caesarean), infants supplemented with bifidobacterium infantis have a complete (on average, more than 70%) dominance of the intestinal microbiome by bifidobacterium longum subspecies of infants. This advantage persists as long as the infant continues to digest breast milk, even after the end of supplementation (day 28), indicating that Bifidobacterium infantis is colonizing the infant's gut at levels above 1010cfu/g feces. Furthermore, the development of those infants which are colonized by Bifidobacterium longum subspecies of infantsThe levels of bacteroides and enterococci, including species of Clostridium (Clostridium) and Escherichia (Escherichia), are much lower.
The microbiome of unsupplemented infants (i.e. infants receiving standard care supported by lactation but not supplemented with bifidobacterium infantis) did not show bifidobacterium infantis levels above 106cfu/g (i.e. limit of detection), and there is a significant difference in microbiome between infants delivered by caesarean section and delivered vaginally. By day 60, 80% (8 of 10) of the unsupplemented infants delivered by caesarean section did not detect bifidobacterial species, whereas 54% (13 of 24) of the vaginally delivered infants did not detect bifidobacterial species. Further analysis of 13 unsupplemented infants in which bifidobacteria could be detected found that these species were mainly bifidobacterium longum subsp. No detectable bifidobacterium longum subspecies of infants was found in any unsupplemented infants in this study. The following examples 1A-1F and international application number PCT/US2017/040530 (incorporated herein by reference) provide further analysis of feces or other characteristics of supplemented and unsupplemented infants.
Example 1A
Metabonomic analysis of infant faeces
Stool samples from the infants of example 1 were evaluated as described below to characterize the stool metabolome and the effects that colonization of this organism may have on the overall metabolism of the infant.
Sample preparation: the fecal samples were kept at-80 ℃ until treatment. Automation using Hamilton Company (Hamilton Company)
Figure BDA0002927731740000311
The system prepares a sample. For quality control purposes, several recovery criteria are added prior to the first step of the extraction process. To remove proteins, small molecules bound to the protein are dissociated or retained in a precipitated protein matrix and chemically diverse metabolites are recovered and the protein is precipitated with methanol under vigorous shaking for 2 minutes (Glen Mills GenoGrinder 2000)And then centrifuged. The resulting extract was divided into five fractions: two fractions were analyzed by two independent Reversed Phase (RP)/UPLC-MS/MS methods using cation mode electrospray ionization (ESI), one by RP/UPLC-MS/MS using anion mode ESI, one by HILIC/UPLC-MS/MS using anion mode ESI, and one sample was kept as backup. The sample is briefly placed in
Figure BDA0002927731740000321
(Zymark) to remove organic solvent. The sample extracts were stored under nitrogen overnight before being ready for analysis.
The study followed (study-tracking) the preparation of the replicates. Small aliquots of each sample were pooled to create study tracking samples, which were then injected periodically throughout the platform run. The variability detected in studies that follow up samples in continuously detected biochemical substances can be used to calculate estimates of overall process and platform variability.
Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS): all methods utilized a Waters acquisition Ultra Performance Liquid Chromatography (UPLC) and seemer femtology (Thermo Scientific) Q-active high resolution/precision mass spectrometer connected to a heated electrospray ionization (HESI-II) source and an Orbitrap mass analyzer operating at 35,000 mass resolution. The sample extract was dried and then reconstituted in a solvent compatible with each of the four methods. Each reconstitution solvent contains a series of standards at fixed concentrations to ensure consistency of injection and chromatograms. One aliquot was analyzed using acidic cation conditions, which were chromatographically optimized for more hydrophilic compounds. In this procedure, the extract was eluted with a gradient of C18 column (Waters UPLC BEH C18-2.1X100mm, 1.7 μm) using methanol and water containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% Formic Acid (FA). The other aliquot was also analyzed using acidic cation conditions, however, it was spectrally optimized for more hydrophobic compounds. In this process, the extract was eluted with the same gradient of C18 column as before using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA, and was run with overall higher organic content. Use ofBasic anion optimized conditions, a separate dedicated C18 column was used to analyze another aliquot. However, the basic extract was eluted from the column with a gradient of 6.5mM ammonium bicarbonate pH 8 using methanol and water. After elution from a HILIC chromatography column (Waters UPLC BEH amide 2.1X150mM, 1.7 μm), the fourth aliquot was analyzed by negative ionization using a gradient consisting of water and acetonitrile and 10mM ammonium formate (pH 10.8). MS analysis data dependent MS in MS and Using dynamic exclusionnThe scans alternate with each other. The scanning range for both methods varies slightly, but covers 70-1000 m/z.
Data extraction and compound identification: the raw data was extracted using proprietary hardware and software, peak qualification and quality control processing. Compounds are identified by comparison to library entries of purified standards or recurrent unknown entities in the library based on validated standards comprising retention time/index (RI), mass to charge ratio (m/z) and chromatographic data (including MS/MS spectral data) for all molecules present in the library. In addition, biochemical identification is based on three criteria: it is recommended to identify retention indices within a narrow RI window, exact mass matching to the library +/-10ppm, and MS/MS forward and reverse scoring MS/MS scores between the experimental data and authentic standards based on a comparison of the ions present in the experimental spectra with the ions present in the library spectra.
Metabolite quantification and data normalization: the area under the curve was used to quantify the peak. For studies spanning multiple days, a data normalization step was performed to correct for variations caused by instrument day-to-day adjustment differences. Essentially, each compound was corrected in a run-day block by registering the median equal to 1(1.00) and scaling each data point (referred to as "block correction"). For studies that do not require more than one day of analysis, no normalization is required except for data visualization purposes.
Determination of the absolute concentration of metabolites in a fecal sample: once the extent of altered metabolites in fecal samples relative to fecal samples taken from infants treated with compositions of the present invention in the hypermetabolic infant fecal samples has been analyzed, a series of known standards are combined to help determine the absolute concentrations of certain metabolites using liquid chromatography-QTRAP or gas chromatography-quadrupole mass spectrometry. A standard curve of known concentrations of metabolites is generated using the identified standards and used to determine the concentration of the metabolite in the fecal sample.
Results
Faecal samples from 20 infants supplemented with bifidobacteria subspecies of infants (intervention) and 20 infants not supplemented (control) were analysed for the levels of 983 metabolites. Analysis of the 983 detected metabolites yielded major findings detailed in international patent application No. PCT/US2018/050973 filed on 9, 13, 2018.
Reduced levels of retinol in the intestinal tract of human infants
An example of a metabolite that is significantly reduced after administration of human milk and bifidobacterium infantis compared to controls is vitamin a (retinol) utilization. Control and infant supplemented with bifidobacterium infantis EVC001 ingested vitamin a from breast milk was approximately the same. However, in faeces, retinol was significantly reduced in infants treated with bifidobacterium infantis EVC001 from example 1.
Elevated levels of gamma-glutamylcysteine and creatinine in the intestinal tract of human infants
Creatinine and gamma-glutamylcysteine, as well as other gamma-glutamyl amino acids, are important for preventing and/or recovering from oxidative stress. Gamma-glutamylcysteine is an important precursor of Glutathione (GSH). It is an important component in the prevention of oxidative stress in mammals. Creatinine is an important metabolite to reduce the effects of oxidative stress and to help prevent oxidative-mediated mitochondrial damage in preterm and high-risk labor. Oxidative stress is a condition that occurs during childbirth. In term infants GSH is usually sufficient, but may not be for premature infants, and GSH may also be lower in people with autism.
Autism is a range of diseases and is best treated early in life to minimize severity. The diagnosis is usually performed after some critical windows are closed. Monitoring the level of oxidative stress during pregnancy and at parturition and recovery from oxidative stress may be an overall indicator of health and may be a tool to minimize the long-term subclinical effects of early oxidative stress by administering the compositions of the present invention.
Non-targeted metabolomic analysis was performed on fecal samples collected on day 28 in example 1 from 20 infants undergoing standard care. The same analysis was done on samples collected from 20 newborn infants receiving the bifidobacterium infantis and human milk oligosaccharide composition in example 1 the relative abundance of glutamyl-dipeptide metabolites was analysed and the results recorded in table 5 below.
Table 3: significant changes in gamma-glutamyl amino acids.
Metabolites Activity/control P-value
Gamma-glutamylalanine 5.9 7.532E-08
Gamma-glutamylcysteine 44.3 1.17E-12
Gamma-glutamyl glutamate 3.1 0.004279002
Gamma-glutamyl glutamine 1.6 0.001325298
Gamma-glutamylhistidine 10.5 6.88631E-05
Gamma-glutamyl isoleucine 8.1 8.144E-07
Gamma-glutamyl leucine 1.6 0.025584145
Gamma-glutamyl-alpha-lysine 3.3 0.052765195
Gamma-glutamyl-epsilon-lysine 2.4 1.66338E-05
Gamma-glutamyl methionine 19.3 8.75E-09
Gamma-glutamylphenylalanine 4.2 5.67663E-05
Gamma-glutamyl threonine 7.15 8.75E-05
Gamma-glutamyl tyrosine 2.8 0.000714584
Gamma-glutamylvaline 3.3 1.11065E-05
Creatinine 1.4 0.021567567
Table 3 shows significant changes in gamma-glutamyl amino acids. Bold values are significant. The p-value is recorded in column 3. Values greater than 1 indicate an increase in intervention compared to the control, while values less than 1 indicate a decrease in intervention compared to the control. The values are activity: ratio of controls or fold change in metabolites produced by treatment. Creatinine and/or gamma-glutamylcysteine may be used as metabolic indicators for monitoring the level of pre-and post-intervention and/or for determining the need for intervention to improve the health status of the infant.
Increased levels of threonine in the human infant gut
Those metabolites that were significantly altered between the supplementation/intervention (Int) and control (con) groups were threonine and its metabolites. Threonine bioavailability was higher in infants fed human milk oligosaccharides and bifidobacterium infantis EVC 001. See international patent application publication No. PCT/US2018/050973, filed 2018, 9, month 13.
TABLE 4 alteration of threonine levels
Int/Con Int/Con, delete outliers
Threonine 3.21 4.35
N-acetyl-threonine 3.07 3.67
Gamma-glutamyl threonine 5.35 7.15
Example 1B
Increased mucin production
In this example, mucin degradation was significantly reduced in EVC001 supplemented infants compared to the control group. The following mucin structures were monitored as part of the metabolome in infant faeces.
TABLE 5 mucin Structure alterations
Figure BDA0002927731740000361
Libraries of known mucin-specific O-glycans were compiled and used to interrogate non-targeted mass spectra of fecal samples. It is hypothesized that modification of the gut microbiome will result in modulation of mucin degradation by gut microbes. The second part of the hypothesis is that colonization by using bifidobacterium infantis (which does not degrade mucin) and subsequent reduction of the mucolytic (mucolytic) group will reduce mucin degradation as measured by the abundance of mucin-specific O-glycans in the infant faeces.
Analysis of the spectra was obtained with nano high performance liquid chromatography-chip/time of flight mass spectrometer (nano-HPLC-chip-TOF MS). Such as Davis et al (2016) ((R))Molecular modulators&Cellular Proteomics15(9): 2987-: e 00501-00517). . Briefly, the HPLC system used was an Agilent 1200 series unit with a microfluidic chip coupled to an Agilent 6220 series TOF mass spectrometer via a chip cube interface. The capillary pump on the chromatograph unit loads the sample onto the 40-nL enrichment column at a flow rate of 4.0 μ L/min and an injection volume of 1 μ L. The nano-pump was used for analyte separation on analytical column, which was 75x43mm and filled with porous graphitized carbon. The separation was accomplished using the method developed for HMO separation using a binary gradient of aqueous solvent a (3% Acetonitrile (ACN)/water (v/v) in 0.1% Formic Acid (FA)) and organic solvent B (90% ACN/water (v/v) in 0.1% FA). The sample was introduced into the TOF mass spectrometer by electrospray ionization, tuned and calibrated using a dual nebulizer electrospray source, with the calibration ions being m/z 118.086-2721.895, and data collected in forward mode. These non-targeted spectra were reanalyzed in the current study.
And (5) analyzing glycan data. Non-targeted mass spectra were collected (as described above) and analyzed using the Agilent MassHunter workstation data acquisition version b.02.01 on a nano HPLC chip/TOF (Frese et al 2017). The "Find Compounds by Molecular characteristics" function of this software was used to identify mucin glycan species within 20ppm of theoretical mass. The abundance of the compounds is expressed as a volume of ion counts corresponding to the absolute abundance of the compounds in each sample. Monitoring 1HexNAc-1NeuAc, 1HexNAc-1Hex-NeuAc, 2HexNAc-1NeuAc, 2He2HexNac-1Hex-1Fuc, 2HexNac-1Hex-1NeuAc, 2HexNac-1Hex-2Fuc, 3HexNac-1Hex-1Fuc, 2HexNac-1Hex-1Fuc-1NeuAc, 2HexNac-1Hex-1Fuc-2NeuAc, 3HexNac-1Hex-2NeuAc and 3HexNac-1Hex-2Fuc-1NeuAc, as they are different human colonic glycans. Robbe et al (2004)Rapid Mass Spectrometry (Rapid report) Communications in Mass Spectrometry)18(4)：412-420。
And (5) carrying out statistical analysis. Multiple t-tests were performed using the Holm-Sidak calibration in Graph Pad Prism 7(Graph Pad Software, la chara, ca, usa). Pearson and spearman correlation tests and principal component analysis were performed in R (v 3.4.2). The AdDossian test (Adonis test) was performed in QIIME 1.9.1 (Caporo et al 2010) using a weighted UNIFRAC distance matrix (Lozupone et al 2011). In the 20 samples analyzed here by nano-HPLC-chip-TOF MS, there was a significant difference in the gut microbiome profile (R) between 10 infants fed bifidobacterium infantis EVC001 and 10 control infants by the adonius test (R)20.62, P < 0.001) (fig. 1). The total Oligosaccharide (OS) composition of the sample was determined by a non-targeted method of nano HPLC-chip-TOF. Compounds were characterized using previously published libraries. Thomsson et al (2002)Sugar biology (Glycobiology)12(1): 1 to 14; robbe et al (2004)Mass Spectrometry flash (Rapid Communications in Mass Spectrometry)18(4): 412-420; matamoros et al (2013)Trends in Microbiology (Trends in Microbiology)21(4): 167-; karav et al (2016)Application and evolution of microorganisms (Applied and environ. micro).82(12): 3622-3630. In these compositions, free HMO (Freese et al (2017) mSphere 2 (6): e00501-e00517) and mucin OS are present in the fecal carbohydrate group of infants. The degradation of human colonic mucin glycans by different gut microbiome profiles was determined as the difference between the microbiome with the predominant bifidobacterium infantis and the microbiome with the more abundant mucolytic group, such as bacteroidetes (bacteroideteceae). As target molecules, 1HexNAc-1NeuAc, 1HexNAc-1Hex-NeuAc, 2HexNAc-1Hex-1Fuc, 2HexNAc-1Hex-1NeuAc, 2HexNAc-1Hex-2Fuc, 3HexNAc-1Hex-1Fuc, 2HexNAc-1Hex-1Fuc-1NeuAc, 2HexNAc-1Hex-1Fuc-2NeuAc, 3HexNAc-1Hex-2NeuAc and 3HexNac-1Hex-2Fuc-1NeuAc were chosen as typical human colonic mucin glycans, e.g., Robbe et al (2004)Mass spectrometry quick report (Rapid Communications in Mass Spectrometry)18(4): 412 and 420.
In FIG. 2, mass spectrometry monitoring of these structures indicated that the number of total OS structures, including isomers and anomers (anomers), in samples from control and EVC001 fed infants ranged from 67.4 + -19.81 and 360.44 + -102.52 (P < 0.001; FIG. 2A), respectively. Although the control sample contained less total OS structure, free human colonic mucin-derived O-glycans of total OS were significantly higher, 25.4(± 8.09); however, only 6.33 (+ -2.24) structures were colonic mucin-derived O-glycans in samples from infants fed EVC001 (P < 0.001, Wilcoxon test; FIG. 2B). As a proportion, the relative abundance of colonic mucin-derived O-glycans in control samples was significantly higher than samples from infants fed EVC001, in terms of number of structures (37.68% + -3.14% and 1.78% + -0.385%, respectively; FIG. 12C, P < 0.001, Wilcoxon test) and the proportion of the total OS profile (26.98% + -8.48% and 1.68 + -1.12%, respectively; drawing (A)2D, P < 0.001, Wilcoxon test).
To examine the interaction of gut microbiome and mucin OS species, pearson correlations were calculated for all groups and structures in the sample, as well as the total abundance and proportion of OS species. The bifidobacterium family (bifidobacterium) abundances were significantly negatively correlated with all mucin core OS species, while Bacteroidaceae (Bacteroidaceae) abundances were significantly positively correlated with 1_1_0_1, 2_1_1_1, 3_1_2_1 and 2_1_2_0 as well as total and number of mucin core OS species (fig. 3). Separately, bacteroidaceae is significantly associated with the percentage of mucin core OS species, with spearman ρ of 0.45(P ═ 0.0393), but bifidobacterium is stronger, but negatively associated with the percentage of mucin core OS species, with ρ of 0.71(P < 0.001). This may indicate the presence of other mucolytic groups in the gut which contribute to or enhance mucin degradation, but which are also negatively associated with the abundance of the bifidobacterium family. Instead, this might indicate that the bifidobacterium family contributes to the consumption of the released mucin OS species. However, this seems unlikely given that bifidobacterium infantis does not degrade mucin as the sole carbon source. In contrast, members of the genus Bacteroides (Bacteroides) distribute most of their genome to harvested polysaccharides, including mucins (Xu et al, 2003), and are significantly positively correlated with mucin OS species concentration release. Many genes involved in polysaccharide utilization have high activity on mucin glycoproteins, including the O-glycan core found in human colonic mucins. Bacteroides can grow on mucin as the sole carbon source and have specific transcriptional responses to incubation with mucin. Marcobal and Sonneburg (2012) Clinical Microbiology and Infection 18(s 4): 12-15. In particular, bacteroides have enzymes from the glycosyl hydrolase families GH 84, GH 85, GH 89, GH 101 and GH 129, which are active on mucosaccharide conjugates.
Example 1C
Improved vaccine response
The infants from example 1 received the vaccine according to a standard schedule. According to the CDC, the standard protocol should look like this (with slightly different schedules in other countries; however, repeated dosing in the first year of life is common).
TABLE 6 CDC vaccine protocol
Figure BDA0002927731740000401
Stool samples collected at 10 and 12 months were used for vaccine quantification to coincide with the appropriate post-vaccination time points and to allow the concentration of vaccine-specific antibodies to expand to plateau after one, two or three doses [ Wright et al (2014) Journal of Infectious Diseases: page 1628-; brown et al (2012) J Immunol Methods: 386(1-2): 117-12]. It was found that the feces in the treatment group showed a significant increase in vaccine-specific antibodies.
EVC001 colonization was evaluated. By titration at baseline and post-feeding as previously described in Freese et al (mSphere 2 (6): e00501-00517)Quantitative PCR uses specific primers for bifidobacterium infantis to measure colonization. When the abundance of Bifidobacterium infantis is more than 105But more preferably greater than 107Or 108CFU/ug DNA, colonization was considered. Colonization can also be described as a significant amplification of the total relative contribution of the bifidobacterium family to the infant's intestinal microbiome.
Microbial production of Short Chain Fatty Acids (SCFA): SCFA can regulate intestinal inflammation by regulating epithelial barrier function. Microbial Short Chain Fatty Acids (SCFA) were extracted by the previously described method and found to be elevated in the treatment group by gas chromatography FID analysis.
Fecal zonulin concentration: zonulin has been described as a major physiological regulator of intercellular tight junctions, and an increased level thereof indicates an increased intestinal permeability. As mentioned before, the concentrations of fecal zonulin were determined using a commercially available ELISA kit (Immundiagnostik, bengham, germany) and found to be significantly reduced in the treatment group.
Fecal TNF expression: TNF is a cytokine that plays a key role in mucosal inflammation and is readily detected at the protein level in children with bowel disease and is found to be significantly reduced in the treatment group.
Uric acid levels of Fatty Acid Binding Proteins (FABPs) and glutathione S-transferase (α -GST): FABPs are small, water-soluble cytosolic proteins that are released into the circulation following loss of the integrity of the intestinal epithelial cell membrane and are therefore markers of gastrointestinal permeability, which have been shown to increase in systemic inflammatory states. Glutathione S-transferases (α -GSTs) are enzymes that are mainly present in liver, kidney and intestinal epithelial cells and are responsible for the detoxification of intracellular endotoxins by binding to glutathione. Plasma (α -GST) levels are peripheral markers of intestinal epithelial cell injury in states of intestinal inflammation and increased permeability. The levels of these biomarkers were assessed using the MILLIPLEX MAP luminex assay (BioRad)) following its standard protocol and were found to be significantly reduced in the treatment groups.
Expression of inflammatory markers (including Toll-like receptor (TLR)2 and TLR4, COX-2 and TNF) in intestinal epithelial cells: previous data have implemented TLR2 in controlling mucosal inflammation by modulating gut epithelial barrier function. Furthermore, a TLR 4-dependent significant increase in Cox-2 expression in intestinal epithelial cells following exposure to lipopolysaccharide has been shown. TNF- α is a cytokine that plays a key role in mucosal inflammation, and its expression and protein levels are readily detectable in children with various intestinal pathologies. As previously described, qPCR was used to determine the expression of TLR2, TLR4, TNF and COX-2 in exfoliated epithelial cells, and was found to be significantly reduced in the treatment group.
Stool microscopy. All samples were fixed for 1-2 hours by adding an equal amount of 5% glutaraldehyde in 0.2M cacodylate buffer (glutaraldehyde at a final concentration of 2.5% in 0.1M cacodylate buffer) and then treated for gram-stain light microscopy and Scanning Electron Microscopy (SEM). For the optical microscope, a sample of conventional gram stain on a slide was imaged using a 60-fold lens and a 2.7-fold optically zoomed EVOS Auto-FL system under the same settings of a color camera. 5 random image fields were collected from each sample slide. For SEM, post-fixation and critical point drying procedures were not performed because the samples were frozen prior to fixation. The fixed sample was dehydrated through an ethanol series and placed on a membrane filter. The samples were mounted on SEM pegs, vented overnight, then vacuum oven dried at 50 ℃ for >2 hours, then sprayed with a thin chromium layer using a Denton Desk V sputter. Images were collected at various magnifications to capture bacterial morphology using a Hitachi S4700 field emission SEM. Microscopic examination confirmed high levels of bifidobacteria in the treated group samples.
Bacterial DNA methods. The relative abundance of fecal bacteria in phyla, class, order, family and genus was characterized by sequence analysis of the V4 fragment of the 16S rRNA gene using QIIME V1.9.1.
Fecal calprotectin. Stool calprotectin levels were quantified using the IDK calprotectin ELISA kit (Immundiagnostik AG, germany) according to the manufacturer's instructions. Absorbance was read at 450nm using a Synergy HT multi-detection microtiter plate reader (BioTek, usa). The samples were double-paneled and tested twice.
Multiplex immunization experiments. Interleukin (IL) -1 β, IL-2, IL-5, IL-6, IL-8, IL-10, IL-22, Interferon (IFN) γ, and Tumor Necrosis Factor (TNF) α were quantified from 80mg feces diluted 1: 10in a Meso Scale Discovery (MSD; Rokville, Md.) kit according to the manufacturer's instructions. Duplicate measurements of standards and samples were set and blank values were subtracted from all readings. The plates were then read on the Sector Imager 24002400 MSD Discovery workbench analysis software. And (5) carrying out statistical analysis. Demographic differences between control and EVC001 fed infants were analyzed using Fisher's exact test for categorical data and verxon rank sum (man-whitney U) test for continuous data. Table 7 shows the data as it is clear from the analysis or visualization. The relationship between fecal calprotectin concentration and% bifidobacteriaceae was quantified using spearman rho correlation, and differences in calprotectin between high (> 25%) and low (< 25%) bifidobacteriaceae were assessed using verxon rank sum test. One subject with abnormally high fecal calprotectin concentrations (greater than 3 standard deviations of the mean of the treatment and control data) was considered to be extremely abnormal and was deleted from the calprotectin analysis described above. Verxolone rank-sum test was performed to assess the relative abundance and cytokine concentration differences of each bacterial population. For radar plots, the median was adjusted to a logarithmic scale and then normalized from 0 to 1 in each cytokine group. Differences in time-dependent changes in each cytokine group were assessed using verxon rank sum test. P values were adjusted using the Bonferonni-Holm method and considered statistically significant if P < 0.05. Statistical analysis was used to determine the significance of the overall cytokine profile, as determined by calculating the Bray-Curtis distance metric, transformed to principal coordinate analysis and displayed using EMPeror. Then, the overall cytokine profile difference divided by group status was determined using permutation-multivariate analysis of variance (PERMANOVA) and significant P-values were determined using 999 Monte Carlo permutation-permutations. To assess the relationship between the overall cytokine profile and the microbiome, we used the Procrustes analysis. The taxonomic manipulation taxonomy unit (OTU) tables at the department level were calculated using QIIME and the respective distance matrices were generated using the cytokine tables, using weighted UniFrac (16S) and Bray-Curtis (cytokines). We performed principal coordinate analysis on both matrices separately and used Procrustes analysis implemented in QIIME to rotate, translate and scale the matrices. The generated transformation matrix is plotted using EMPeror. P-values for Procrustes analysis were generated using Monte Carlo simulations (n-999). By calculating the P-value using Fisher's Z-transform, the raw correlation statistics specify the likelihood of these associations as true positive correlations to normalize the distribution of the correlation scores.
Figure BDA0002927731740000431
Results
Demographics and stool analysis of infant participants. To investigate the effect of gut microbiome on host immune response, we used fecal samples from 20 controls and 20 EVC001 feedings at postnatal days 20, 40 and 60. Subjects of EVC001 and control did not differ in the selection of randomized parameters; however, the mother of the control infant is more likely to be the first time to become a mother (P < 0.01) and younger (P < 0.01) than the mother whose infant randomly received Bifidobacterium infantis EVC 001. There were no significant differences between the two groups in time of delivery, mode of delivery, use of antibiotics during delivery, gestational age, sex, weight at delivery or discharge, length of birth, maternal BMI or weight gain in pregnancy or maternal GBS diagnosis.
Infants fed bifidobacterium infantis EVC001 significantly increased the abundance of the bifidobacterium family in the intestinal microbiome of infants. We first evaluated microbiome profiles of both groups at day 6 (baseline), day 40 and day 60 after birth (table 8). There were no statistical differences between the two groups of infants included in this prospective study in the four main representative groups (Bifidobacteriaceae, Bacteroidaceae, Bifidobacteriaceae, Clostridiaceae and Enterobacteriaceae), which could be identified on day 6 after birth, before the start of supplement addition (fig. 4). On day 40, the abundance of bifidobacterium family in the group fed bifidobacterium infantis EVC001 was significantly higher (P < 0.0001) compared to the control (fig. 4). In contrast, in infants receiving EVC001 treatment, the abundance of bacteroidaceae and clostridiaceae groups was significantly reduced, while enterobacteriaceae groups were very significantly reduced (P < 0.05, P < 0.05 and P < 0.0001, respectively) (fig. 4). Similarly, at postnatal day 60 (32 days after the last feeding with EVC001), infants fed EVC001 had a higher abundance of bifidobacteriaceae in the microbiome compared to control infants, while bacteroidetes and clostridiaceae were less abundant (all P < 0.0001) (fig. 4).
Figure BDA0002927731740000441
Light microscopy and scanning electron microscopy were used on three fecal samples from the day 40 control and EVC001 feeding groups, respectively. Gram staining showed that the fecal smears of the control group contained predominantly gram negative bacteria, while the vast majority of samples from infants fed EVC001 contained gram positive bacteria. Multiple fields of view of control fecal samples determined several distinct bacterial morphologies, while samples from infants fed EVC001 showed a uniform rod-like bacterial morphology with few longitudinal divisions, consistent with our molecular observations.
Fecal calprotectin levels are directly related to the abundance of the bifidobacterium family. Malnutrition, including low abundance of bifidobacteriaceae in the infant gut, is associated with increased inflammation. To determine whether bifidobacteria abundance in the gut correlates with gut inflammation in our group, stool collected from postnatal day 40 was analyzed using spearman correlation and the concentration of fecal calprotectin, a marker of gut inflammation, was compared to the abundance of bacterial populations. Data observed from day 40 showed that there was a significant correlation between bifidobacteria abundance and lower fecal calprotectin levels (r)s-0.72, P < 0.0001; fig. 5 a). These data also provide a clear bimodal distribution in which the abundance of bacteria of the bifidobacterium familyDegree ≦ 25% was considered to be hypobifidobacteriaceae, whereas > 25% indicated to be hyper bifidobacteriaceae (FIG. 5 a). Samples containing low levels of bifidobacterium showed a significant increase in intestinal inflammation (measured by fecal calprotectin) compared to samples containing high levels of bifidobacterium using 25% abundance cut-off values (P < 0.01; fig. 5 b).
Colonization by bifidobacterium infantis EVC001 is associated with a reduced expression of pro-inflammatory cytokines in stool. Cytokine profiles at day 6, day 40 and day 60 post-natal were evaluated. At baseline, IL-1 β concentrations were significantly higher in the control (P < 0.05; FIG. 6a) compared to EVC001 infants; however, there were no other statistically significant differences between the two groups at baseline. On postnatal day 40, there was significant modulation of fecal cytokine profiles in infants fed EVC001 compared to controls. In particular, IL-8, IL-22, IL-1. beta. concentrations were significantly lower in EVC001 infants compared to control fecal samples (all P < 0.01; FIG. 6b) and IFN. gamma. (P < 0.001; FIG. 6 b). This trend continued until day 60, during which time the control group produced significantly higher levels of IL-6, IL-22, TNF α, IL-1 β and IFN γ (P < 0.01, P < 0.05, P < 0.01 and P < 0.05, fig. 6c) compared to infants in which bifidobacterium infantis EVC001 was colonized. Taken together, these data show major overall cytokine differences between infants not fed EVC001 on the first 60 days of birth compared to pure breast-fed infants fed EVC001 (table 9).
Figure BDA0002927731740000451
Control infants showed significant decreases in IL-2 and IL-5 from day 6 to day 40 (P < 0.05 and P < 0.01; FIGS. 7a, b and i); however, the control showed significantly higher levels of IFN γ from day 6 to day 40 (P < 0.0001). Furthermore, there was no significant difference in cytokine levels in the control infants from day 40 to day 60 (fig. 7 a-i); however, IL-22 and IFN γ were significantly higher in day 60 fecal samples compared to day 6 fecal samples (P < 0.01, P < 0.0001; FIGS. 7f and i, respectively).
In contrast, infants fed EVC001 produced significantly lower levels of IL-2, IL-5, IL-6, IL-10, IL-22, TNF α; however, IFN γ was significantly increased on day 40 compared to day 6 (all P < 0.0001; FIGS. 7 a-f). Furthermore, IL-22 at day 60 was significantly higher than at day 40 (P < 0.05; FIG. 7 f). Fecal levels of IL-2, IL-5, IL-6, IL-10 and TNF α were significantly lower at day 60 compared to day 6 (all P < 0.0001; FIGS. 7a-c, e, g), while IFN γ levels were significantly higher at day 60 compared to day 6 (P < 0.0001; FIG. 7 i).
These results show the major difference in cytokine profile in the first 60 days of life between EVC001 fed infants and control infants. Most notably, fecal cytokine levels were significantly lower and remained low in infants receiving EVC001 during the first 60 days after birth, whereas fecal cytokine levels of control infants varied depending on the cytokine, but overall cytokine levels increased from day 6 to 60 after birth (table 10).
Figure BDA0002927731740000471
Bifidobacterium infantis EVC001 colonization affects the cytokine profile. To identify the major drivers of measured fecal cytokines, we used Principal Component Analysis (PCA) as a dimension-reduction technique, using all of the parameters of the above clinical data, proinflammatory cytokine concentrations and panels. By adding clinical data, the cytokine profile composition did not differ in infants on day 6 before receiving EVC001, as shown by PCA of day 6 stool samples (fig. 8 a); however, by day 40 after birth, there was a distinct cluster difference between the EVC001 fed group and the control group (P0.001, pseudo-F12.5; fig. 8 b). Such differentiation was still evident at day 60 and had more pronounced clustering compared to earlier time points (P0.001, pseudo-F13.9; fig. 8 c). These observations confirm that colonization and timing of bifidobacterium infantis EVC001 is a major factor affecting differences in fecal cytokines.
There is a significant correlation between gut microbial abundance and inflammatory bowel cytokine response. We performed pairwise correlation tests between microbiologic composition and specific cytokine concentrations detected in feces of purely breast-fed infants on postnatal day 6 (baseline) and postnatal days 40 and 60 (spearman correlation α < 0.02 corrected using Benjamini-Hochberg FDR). A total of four groups were found to be significantly associated with specific proinflammatory cytokines, including clostridiaceae, enterobacteriaceae, Peptostreptococcaceae, and Staphylococcaceae. Specifically, the clostridiaceae family is significantly associated with IL-1 β, IL-8, IFN γ, and TNF α production at postnatal day 40, and IL-1 β, IL-6, IL-8, IL-22, IFN γ, and TNF α production at postnatal day 60. Enterobacteriaceae is significantly associated with increased levels of IL-1 β, IL-8, IL-22, IFN γ, and TNF α at postnatal day 40, and increased levels of IL-1 β, IL-6, IL-22, IFN γ, and TNF α at postnatal day 60. Consumption of Streptococcus peptidoglycan was significantly associated with IL-22 and TNF α at day 40, while the family Staphylococcus was associated with increased IFN γ concentrations at day 40. Furthermore, 5 proinflammatory cytokines (IL-1 β, IL-8, IL-22, IFN γ, and TNF α) were found to be negatively associated with bifidobacteria at postnatal day 40, and 6 proinflammatory cytokines (IL-1 β, IL-6, IL-8, IL-22, IFN γ, and TNF α) were found to be negatively associated at postnatal day 60 (FIG. 9).
Evaluation of secretory IgA in infant faeces. sIgA of stool samples from example 1 was quantitatively measured in vitro by a sandwich enzyme immunoassay (RedBelot, CA; https:// www.reddotbiotech.ca/files/manuals/5c63251c-5b44-4771-b920-55 e8d8bo5a9. pdf). The results were then correlated with the relative abundance of the bifidobacterium family by 16s DNA sequencing. The results in fig. 10 show that sIgA increases with increasing bifidobacteria, with spearman p 0.40 and p 0.0389. This is used as an example only and is not intended to limit the use of the different compositions outlined herein to increase the bifidobacteriaceae family or different study design to assess the benefit of increasing sIgA by those skilled in the art.
Evaluation of IL-17, IL-4 and IL-13. The difference in IL-17 between control infants and EVC001 treated infants can be used as a measure of malnutrition (fig. 11).
The stool was evaluated for antigen specific response in infants colonized with bifidobacterium infantis EVC 001. Biophysical antibody profiling experiments were performed to assess the Fv and Fc characteristics of vaccine-elicited antibodies in infants with and without colonization by bifidobacterium infantis. First, fluorescently encoded magnetic microspheres were functionalized with the antigens listed in table 11. Other antigens may be suitably taken into account or exchanged with those listed in table 11. The test samples were evaluated for the presence and phenotype of antibodies by staining the bead-bound antibodies with PE-coupled detection reagents (anti-IgG, anti-IgA, and potentially anti-IgE) and detection via flow cytometry. For this test, 100 μ L of each stool sample was prepared from 34 subjects (2 time points, up to 62 samples in total) who received EVC001 or belonged to the control group.
Figure BDA0002927731740000491
It was observed that infants fed bifidobacterium infantis EVC001 and continuously or permanently colonized with bifidobacterium infantis at least the first 100 days of life showed a stronger vaccine response than infants without bifidobacterium infantis colonization.
Example 2
Expanding the Treg population of mice
Germfree mouse pups were lactating and fed a polysaccharide-free mouse diet containing vitamin a at 3 or 4 weeks after birth. Once the mouse pups were weaned, the dysbiosis (bifidobacteria-free and hypervariable phyla) human infant microbiome was drenched on experimental day 1. Mice were divided into 4 groups: control group (placebo: placebo); control (bifidobacterium infantis placebo) plus LNnT; bifidobacterium infantis plus LNnT placebo; and bifidobacterium infantis plus LNnT. All groups received bifidobacterium infantis or placebo by drench every 3 days. LNnT or placebo was added to the drinking water for 21 days. At the end of the supplementation period, mice were necropsied. The spleen and caecum weights were greater in mice receiving bifidobacterium infantis plus LNnT, consistent with immune cell expansion (fig. 12A and 12B). Blood leukocytes were collected and populations of T regulatory cells, Th17 cells, CD4, CD8 cells from various sources were characterized and evaluated using flow cytometry for: CD4, CD25, Foxp3, Helios, neuropilin, CD8, CD44, CD62L, interferon Y (IFN γ), interleukin 17(IL-17), transforming growth factor β (TGF β), interleukin 35(IL-35), CD24, CD27 and/or CD38 (FIGS. 13A-F depict an analysis of lymphocyte populations in untreated and treated mice (A) total lymphocytes, (B) CD4+ (C) CD4+/CD25+ Helios-FoxP3+, (D) CD4+/CD25 +/ioHelos-FoxP 3-, (E) CD4+/FoxP3+ CD25-, (F) CD4+/Helios + CD25-, plasma cytokines and innate immune factors are evaluated daily, and faecal colony formation of infants is assessed using PCR and a colony forming method for colon colony forming and colon colony forming For histopathology, qRTPCR and proteomics. Mice receiving bifidobacterium infantis plus LNnT were found to have a greater number of naive B cells for antigen presentation. They were also found to have a thicker mucus layer on the epithelial surface and more CD4+ FoxP3+ T regulatory cells than mice that remained dysbiosis.
Example 3
Prevention of rat peanut allergy
Mouse pups born under sterile conditions were lactated 3-4 weeks after birth and fed a polysaccharide-free diet rich in vitamin a. Once the mouse pups were weaned, the dysbiosis (bifidobacterium free species and proteobacteria hyperblastomycota) human infant microbiome was drenched on experimental day 1. Mice were divided into 4 groups: control group, control plus LNnT, bifidobacterium infantis and bifidobacterium infantis plus LNnT. The composition was administered to mice for 21 days. All groups received bifidobacterium infantis or placebo by drench every 3 days. LNnT or placebo was added to the drinking water for 21 days. Mice received intragastric drench of peanut extract 5 mg/mouse per week for sensitization, and then 25 mg/mouse at week 5, challenged with 10 μ g cholera toxin to elicit an allergic response. Control animals received intragastric drench vehicle only. Symptoms of anaphylaxis were assessed and mice were necropsied. Plasma samples were collected for evaluation of histamine and IgE concentrations. Blood leukocytes were isolated for PBMC characterization and evaluation of regulatory T cell population expansion using flow cytometry specific for: CD4, CD25, Foxp3, Helios, neuropilin, CD8, CD44, CD62L, IFN γ, IL-17 and B cell populations, including Breg and plasma cells, using the following markers: IgM, CD5, CD24, CD19, CD19, CD20, CD34, CD38, CD45R, CD78, CD80 and CD 138.
Administration of bifidobacterium infantis and subsequent colonization of the mice with bifidobacterium infantis was found to reduce the inflammatory response to food antigens compared to control mice. Colonization by Bifidobacterium infantis will prevent allergic reactions and/or alleviate symptoms of allergy such as core body temperature drop, increase in serum allergen-specific IgE and IgG1, IL-4 and MCPT1, mast cell expansion in the jejunum, increased IL-13 production, edema and mast cells, eosinophils and/or dendritic cell expansion, increase in IL-4 secreting CD4+ T cells in Mesenteric Lymph Nodes (MLN) and spleen, decrease in the number of Foxp3+ Tregs in colon, spleen and/or MLN, and allergic diarrhea.
Example 4
Reversal of rat peanut allergy
Mouse pups born under sterile conditions were lactated and fed a polysaccharide-free mouse diet 3 weeks after birth. The dysbiosis microbiome of example 3 was drenched on experimental day 1. Mice received intragastric drench of peanut extract at 5 mg/mouse on days 1 and 7, then 25 mg/mouse on week 5, and 10 μ g cholera toxin. The initiation of the reaction was carried out at 8-10 weeks using intragastric instillation of 10 mg/mouse food antigen (e.g., peanut extract) and vehicle (PBS). At week 10, the test group of mice was fed with bifidobacterium infantis, while the control mice received only a vehicle drench. All mice were free to eat drinking water infused with LNnT over a 21 day period. Mice were challenged again with food antigen by intragastric drenching with 10 mg/mouse peanut extract and vehicle (PBS). Symptoms of anaphylaxis were assessed and mice were necropsied. Plasma samples were collected for evaluation of histamine and IgE concentrations. Blood leukocytes were isolated for PBMC characterization and evaluation of expansion of regulatory T cell populations using flow cytometry specific for CD4, CD25, Foxp3, Helios, neuropilin, CD8, CD44, CD62L, IFN γ, IL-17. Administration of bifidobacterium infantis and subsequent colonization of the mice with bifidobacterium infantis reduced the inflammatory response to food antigens in animals that previously showed sensitivity to food antigens compared to control mice.
Example 5
Prevention of human peanut allergy in infants
Infants at 2-4 months of age at risk of allergy were recruited and screened for bifidobacterium infantis status (bifidobacterium infantis abundance) in their faeces. Infants were divided into 2 groups: bifidobacterium hyperlongum (ideal infant gut microbiome) and Bifidobacterium oligoum (dysbiosis infant gut microbiome). If Bifidobacterium levels in the Bifidobacterium bifidum-deficient stool sample are below the threshold 10 as measured by next generation sequencing, such as 16S RNA sequence measurement8CFU/μ g DNA and/or bifidobacterium family is less than 35% of the total infant intestinal flora and the bifidobacterium bifidum stool sample is determined to be bifidobacterium bifidum or dysbiosis. In contrast, the Bifidobacterium hyperbifidum sample or ideal infant gut microbiome threshold is 108CFU/μ g DNA or higher and/or bifidobacterium family is more than 35% of the total infant intestinal microbiome. Once the infants were screened, the dysbiosis group was randomized into placebo or supplementary group, which received activated bifidobacterium infantis, LNnT and retinol. The infant was fed either supplement or placebo for 16 weeks. Peanut extract was introduced into the diet for 3 days at 12 weeks for infants at least 4 months old. Blood samples were collected at 12, 16, 20, 24, 36, 40 weeks for analysis of TReg cells and IgE. The infants were taken back at 9 months of age and subjected to peanut challenge. Allergy events between groups were analyzed. Improved tolerance of peanut extract was identified in the bifidobacterium infantis and LNnT groups compared to the dysbiosis group, resulting in reduced allergic reactions, including a significant reduction in the concentration of specific IgE of peanut extract.
Administration of bifidobacterium infantis and subsequent colonization of bifidobacterium infantis in infants reduces the inflammatory response to food antigens compared to control groups with low levels of colonization.
Example 6
Reversion of peanut allergy in children younger than 3 years of age
9-18 month old peanut allergic infants were recruited to participate in an immunotherapy regimen to reverse their known peanut allergy. Infants eat a diet containing oligosaccharide dietary components including 15 grams/day of a formulation comprising 50% LNnT and 25% LNT and 25% GOS, and a supplement of bifidobacterium infantis (40 billion CFU per serving) twice daily, and a source of dietary β -cryptoxanthin, such as orange, to consume 12 mg/day (the expected yield of retinol is 500 μ g/day) for 12 weeks. At week 8, a low dose of peanut extract was added to the diet for 1 week under medical supervision. At week 16, the infant was brought back for peanut challenge.
Administration of bifidobacterium infantis and subsequent colonization of the infant by bifidobacterium infantis may reduce the inflammatory response to food antigens.
Example 7
Prevention of Atopic progression (Atopic March)
Infants were enrolled at birth and randomized into 4 groups: 1) a placebo; 2) bifidobacterium infantis EVC001 and a dedicated breast milk diet; 3) bifidobacterium infantis EVC001 and fed with a proprietary formula containing 8g/L LNT; 4) bifidobacterium infantis EVC001 and fed with a proprietary formula containing N-glycans released from 8g/L bovine whey proteins. Infants are fed Bifidobacterium infantis EVC001 until they stably colonize > 10in 100 days of life6. After 100 days, the infants continue to follow the same feeding strategy as at the beginning, up to 6 months of age, without supplementation with bifidobacterium infantis EV 001. Stool samples were collected once a week during the study, and blood samples were collected once a month during the study. After a 100 day high bifidobacterium infantis period, follow-up visits were made in the next year. Stool samples were analyzed for metagenomics, metabolomics, qPCR sIgA, and stool cytokines. The blood sample analysis will include: immune cell characterization,Cell function analysis, metabolomics, epigenetics, metagenomics, innate immunity and acute phase protein quantification, cytokines (especially IL-4 and IL-13), and IgG1 and IgE antibody quantification (including autoantibodies). The results may include one or more of the following: improved Treg/Th17 ratio compared to placebo; increase Treg cells or decrease Th17 cells; increase the number of B regulatory cells, reduce cytokine production, reduce the level of innate immune factors, reduce acute phase protein release; increasing vaccine response or potency, reducing autoantibodies and/or reducing inflammation.
The therapeutic outcome includes a reduction in atopy, asthma, eczema. Reducing the incidence of atopic diseases, including atopic wheezing, asthma. Other autoimmune and inflammatory disorders can be assessed, such as type I diabetes, inflammatory bowel disease during the subsequent follow-up period until the age of 6 years of life.
Example 8
Prevention of type I diabetes in mice
Germfree born non-obese diabetic (NOD) mouse pups milk and eat a polysaccharide-free mouse diet containing vitamin a at 3 or-4 weeks after birth. Once the mouse pups were weaned, the dysbiosis (bifidobacterium free species and proteobacteria hyperblastomycota) human infant microbiome was drenched on experimental day 1. Mice were divided into 4 groups: control group, control plus LNnT, bifidobacterium infantis and bifidobacterium infantis plus LNnT. The composition was administered to mice for 21 days. All groups received bifidobacterium infantis or placebo by drench every 3 days; LNnT or placebo was added to drinking water. Additional mice were divided into 2 groups. On day 21 of the experiment, these groups were drenched with the healthy human infant microbiome (bifidobacterium species). Mice were then fed placebo or LNnT in their drinking water and received vitamin a in their diets. Mice were monitored for diabetes by weekly tail vein blood glucose measurements and read > 14mmol on two consecutive days-1Then euthanized. At necropsy, lamina propria, spleen, mesenteric lymph nodes and blood samples were collected for PBMC characterization and evaluation of expansion using flow cytometry on CD4, CD25, Foxp3, Helios, neuropilin, CD8, CD44, CD62L, IFN γ, IL-17, IgM, CD3, CD5, CD24, CD19, CD19, CD20, CD34, CD38, CD45R, CD78, CD80 and CD138 are specific. Plasma was evaluated for cytokines and innate immune factors. Fecal samples were collected daily and fecal microbial colonization was assessed using qPCR and/or 16s and/or shotgun sequencing. Ileum and colon were collected for histopathology, mucin content qPCR and proteomics.
Administration of bifidobacterium infantis and subsequent colonization of the mice with bifidobacterium infantis will reduce the incidence of diabetes development compared to control mice.
Example 9
Evaluation of the effects of Bifidobacterium infantis on the development of auto-immune and/or allergic diseases (T1D) in breast-fed infants Studies on nutritional intervention
Emerging evidence suggests that over the past 50 years, breast-fed human infants have reduced bifidogenic colonization of infants, partly due to antibiotic use and a decrease in breast-feeding rates. The lack of bifidobacterium infantis colonization during this period in infants is associated with an increased incidence of childhood autoimmunity (e.g. T1D, celiac disease) and allergic diseases (e.g. eczema, asthma). It is speculated that restoring a high proportion of colonization of the major intestinal tract by bifidobacterium infantis in infants may improve Treg cell levels and/or activity and reduce the incidence of autoimmune and allergic disease in childhood.
Administration of activated bifidobacterium infantis to breast-fed newborns and infants consistently results in high levels of bifidobacterium infantis in the faeces. This study will investigate whether inducing bifidobacterium infantis colonization of breast-fed infants with bifidobacterium infantis EVC001 would reduce the risk of developing T1D, as measured by seroconversion to various islet cell autoantibodies (e.g., stage 1T 1D) at 36 months of age.
This is a randomized, double-blind, placebo-controlled, parallel group study design (see fig. 15). After the parent/guardian signs the informed consent, the infant will be screened for all eligibility criteria and if found to be eligible, will be included in the study. Eligible infants must first be genotyped for HLA collected from cord blood samples. They will be classified into group 1 (low risk of HLA genotype) and group 2 (high risk of HLA genotype) according to their HLA genotype. Groups were randomly assigned as either placebo or treatment groups. After collecting baseline assessments, infants were randomized to receive either bifidobacterium infantis EVC001 or placebo once a day for 12 months. Parents/guardians of the infants will be instructed to remain breastfed (at least 1 feeding per day) for at least 6 months, if possible 12 months, during the treatment period. After a 12-month treatment period, the individuals will be periodically subject to study visits and assessments. The individuals were followed until a sufficient number of events (seroconversion to multiple (. gtoreq.2) islet autoantibodies) had accumulated to obtain the planned work force (planed power) for the final analysis.
Schematic overview of the study
Participants received bifidobacterium infantis EVC001 or placebo daily for a total of 12 months, which would be delivered by the parent/guardian or other caregiver at home. Single dose sachets (containing 80 billion CFU of activated bifidobacterium infantis EVC001+ lactose) or matched placebo sachets are administered daily. Upon administration, a single bag of lactobacillus infantis EVC001 or placebo is mixed with a few spoons of expressed breast milk and then delivered to the infant's mouth at the beginning of feeding.
Individuals who found seroconversion to multiple autoantibodies during the course of the study will be monitored regularly for evidence of asymptomatic blood glucose abnormality (stage 2T 1D) and symptomatic blood glucose abnormality (stage 3T 1D). During the course of the study, any participants found to have stage 2 or stage 3T 1D were monitored and treated according to standard of care.
The total treatment time for all groups will be 12 months. All mothers (not in the subgroup) will be encouraged and supported to continue breastfeeding for at least 6 months, if possible, for the entire 12-month treatment period.
And (3) evaluating the efficacy:the primary efficacy assessment would be seroconversion of >2 out of 4 islet autoantibodies (IAA, GAD65, IA2 and ZnT 8). Secondary efficacy assessments will include the frequency of eczema and infant colicAnd (4) rate. Exploratory efficacy assessments included body weight changes and seroconversion positive for tissue transglutaminase autoantibodies.
Biomarker assessmentSerum/plasma IgE-sum antigen specificity (cat, dog, egg, milk, house dust mite, tail grass, birch and peanut), fecal microbiome analysis (shotgun metagenomics) and fecal metabolomics will be included.
Other evaluationIncluding DEXA scanning of body composition, health questionnaires to follow breastfeeding, sleep patterns and symptoms of colic, disease screening questionnaires to collect signs of development of eczema, allergic rhinitis, asthma or other allergic diseases, and skin prick tests on a panel of allergens.
Blood samples were collected at baseline, 3, 6, 9 and 12 months post-partum for assessment of peripheral blood mononuclear cell characteristics (including regulatory T cells), islet cell autoantibodies, vaccine response.
Infants colonized with bifidobacterium infantis increase TReg cell numbers and reduce the probability of autoimmune and allergic diseases in childhood.
Example 10
Assessment of Bifidobacterium infantis on autoimmune and/or allergic diseases in infants fed with infant formula (T1D) Nutritional intervention study of the impact of development
To determine the effectiveness of altering the development of T1D in infants, the study design of example 8 was modified to recruit infants between 0-1 month of age fed exclusively with infant formula. These infants are fed a composition comprising LNT, activated bifidobacterium infantis, vitamins a and D. MCT oil is administered daily, together with vitamins a and D and a single composition of bifidobacterium infantis, for the first 6 months of life. The oil portion is added to a small amount of reconstituted infant formula and administered as a single serving. The total LNT intake was calculated from the daily concentration of 12 g/L. The LNT supplement is packaged to provide the appropriate amount of LNT per 2 ounces of infant formula, and each bottle receives a dose of LNT according to the volume required (i.e., for an 8 ounce bottle, 4 sachets of LNT would be used). The effect of the treatment was evaluated at 2 months, 6 months, 12 months, 18 months, 24 and 36 months.
It will be appreciated by those skilled in the art that examples 8 and 9 provide options for breast feeding and infant formula feeding, and that the exact schedule of supplements and compositions may be modified to other embodiments described herein as part of the invention. These specific examples are for illustration purposes only. In other examples, one may study LNT and vitamin a supplements compared to placebo.
Example 11
Improved vaccine response in IMMUNOSENESCENT (IMMUNOSESCENT) individuals
The elderly are screened for antibody titers against past pneumonia vaccines. High risk groups (low antibody titer/low immune function) were divided into 3 groups: placebo control group and group receiving threonine containing protein, vitamin A, LNT/GOS and bifidobacterium infantis combination, and group receiving vitamin a and LNT. The individual took the supplement once daily for a total of 8 weeks (4 weeks before vaccination and 4 weeks after vaccination). At week 4, subjects were given a DTap vaccine. Antibody titers were evaluated 4 weeks and 3 months after vaccination.
Adults receiving a composition comprising bifidobacterium infantis, LNT and vitamin a are expected to show higher antibody titers than adults in the placebo control group and may also be compared to LNT and vitamin a alone.
Example 12
Prevention and/or treatment of SAM
Malnutrition is a problem that persists worldwide. It is estimated that children under the age of 5, over 1800 million, are affected by the most extreme form of malnutrition, namely Severe Acute Malnutrition (SAM). Children with SAM are more than 12 times more likely to die than well-nourished children. Infectious morbidity is prevalent among survivors. The causes of malnutrition are generally thought to be associated with chronic poverty, lack of nutritional foods, lack of proper breast feeding, recurrent infections and poor hygiene. While it is possible to improve the condition of malnourished children by providing them with adequate nutrition, there are still groups that are not amenable to current therapeutic intervention. Studies have shown that gut microorganisms are associated with malnutrition and that children with SAM have gut dysbiosis that will mediate some of the pathological conditions of their disease. The standard of care for these children should be enhanced by interventions that correct intestinal dysbiosis, barrier function and B and T cell function, improve weight gain during nutritional rehabilitation and reduce the incidence of infectious diseases.
In a single-blind RCT stratified randomized study, the measured dry prognosis will modulate T cell responses in the infant's intestine 28 days later by administration of bifidobacterium infantis, LNnT and a nutritional supplement containing vitamin a (figure 15). The target population was first divided into 2 groups as shown in fig. 2. Group 1 consisted of SAM infants with severe acute malnutrition at 2-6 months of age, randomly assigned to three treatment groups after completion of the stabilization phase of SAM treatment; SAM will be defined as height relative to body weight < -3Z. Group 2 consisted of non-malnourished infants (WLZ ≧ -1) hospitalized for < 6 months of age who received antibiotic treatment for infection. The infants in this group will receive at least 50% of the nutritional intake from breast milk to meet the group entry criteria. Exclusion criteria (for both groups): septic shock or very severe pneumonia, need for assisted ventilation or acute kidney injury when admitted, congenital defects that prevent feeding, such as cleft palate, chromosomal abnormalities, jaundice, tuberculosis, bilateral pedal edema, and antibiotics (currently used antibiotics) for breast-fed infant mothers. Other exclusion criteria for group 1 (SAM) infants received > 75% nutrition from breast milk. Other exclusion criteria for group 2 (non-malnutrition): infants who receive < 50% nutrition from breast milk.
Microbiome response of the patient population group 1 to probiotic supplements (with and without prebiotics) was monitored to demonstrate a larger scale clinical outcome study. In addition, non-malnourished infants hospitalized for infectious conditions face challenges related to malnutrition caused by antibiotic use. In group 2, the ability of activated bifidobacterium infantis EVC001 to rescue the microbiome of predominantly breast-fed non-malnourished infants will be evaluated.
Fecal samples were collected for assessment of bifidobacterium infantis colonization as well as markers of mucosal epithelial monolayer integrity and inflammation. Blood samples were collected at baseline and 28 days for assessment of peripheral blood mononuclear cell characteristics (including regulatory T cells, B cells and plasma cells) and vaccine response. The infants are assessed for symptoms or development of severe acute malnutrition or intestinal infection, including sepsis.
Children receiving bifidobacterium infantis showed improved B cell and plasma cell characteristics, vaccine response, better weight gain, better lean to fat mass ratio, TReg cell expansion and a reduced incidence of acute malnutrition symptoms.
Example 13
In vitro identification of antigen recognition sites on Bifidobacterium infantis important for tolerance and TREG cell expansion
To determine the important cell surface components required for increased tolerance of bifidobacterium infantis upon interaction with host immune cells, we assessed the exopolysaccharides, proteins and/or genes involved in the interaction between the bifidobacterium infantis cell surface and dendritic cells to initiate development/expansion of the TReg cell population.
Step 1. identification of Extracellular Polysaccharide (EPS) secretion in activated Bifidobacterium infantis
EPS secretion of activated bifidobacterium infantis will be determined by electron microscopy according to the procedure from Schiavi et al (2016) AEM: 82: 7185. Bifidobacterium infantis cells were grown for 48 hours on liquid medium without yeast extract of MRS containing lactose (non-activated cells) or human milk oligosaccharides (activated cells) as sole carbon source. After culture in MRS medium, the bacteria were gently rinsed in PIPES (piperazine-N, N-bis-2-ethanesulfonic acid) buffer (0.1M, pH 7.4) and fixed in 2.5% glutaraldehyde resuspended in PIPES buffer for 5 minutes. The samples were rinsed twice in PIPES buffer (2 min each) and postfixed with 1% osmium tetroxide in 0.1M PIPES buffer (pH 6.8) for 60 min in the dark. The samples were then washed 3 times in milliq water (2 minutes for each wash) and then dehydrated through a series of ethanol (50, 70, 96 and 100%), steps 5 minutes each. All fixation and washing steps were performed at room temperature. After dehydration, the sample was then critical point dried and coated with 10nm gold/palladium (80/20). The bacterial preparation was examined using a Scanning Electron Microscope (SEM).
Step 2. extraction and quantification of Exopolysaccharide (EPS) secretion in activated Bifidobacterium infantis
To determine EPS secretion in bifidobacterium infantis, cells were grown for 48 hours on agar plates without yeast extract of MRS comprising lactose (non-activated cells) or human milk oligosaccharides (activated cells) as sole carbon source. After 48 hours, EPS was extracted, with some modifications, according to Altmann et al 2016. Briefly, cells were resuspended in phosphate buffered saline and mixed with three volumes of cold absolute ethanol to a final concentration of 80% (v/v), then precipitated overnight in ethanol solution at 4 ℃. The precipitate was removed with a spatula and resuspended in miliQ water. Purification of contaminants and residual ethanol can be performed on a C18 filter cartridge connected to a vacuum manifold. The eluted EPS was filtered through a 0.45 μm syringe filter and quantified by phenol-sulfuric acid colorimetry at 490 nm as described by Matsuko et al 2005. The level of secreted EPS in activated and inactivated bifidobacterium infantis was quantified in nmol/well.
Step 3, characterizing the composition of Exopolysaccharides (EPS)
The EPS composition will be elucidated following the method of Gonzalez-Gil et al 2015 and using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with modifications. The sample containing EPS was mixed in a ratio of 1: 1 with 20-40mg/ml of a matrix of 2, 5-dihydroxybenzoic acid (DHB) dissolved in 30% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in milliQ water. A volume of 2u1 of the sample/matrix mixture was then spotted on a MALDI target plate for analysis. The spectra were compared with reference polysaccharides (dextran, gellan gum, xanthan gum and alginate) prepared in the same way as the experimental samples. MALDI-TOF MS was run in forward mode as recommended by the manufacturer for polysaccharide characterization.
Step 4. characterization of solute binding proteins and other signaling molecules in activated and unactivated cells
In addition to EPS, proteins and signal molecules (e.g., glycolipids) will also be analyzed for secretion into the extracellular space. Bifidobacterium infantis cells were grown for 48 hours in 500ml MRS containing lactose (non-activated cells) or human milk oligosaccharides (activated) as sole carbon source. Cell yields were normalized by optical density and centrifuged at 6012 Xg. The supernatant was then filtered through a 0.2 μm syringe filter and divided into 2 fractions. One part will be used for protein collection and the other part will be used for lipid and glycolipid identification. In the first fraction, the proteins were concentrated using Amicon ultra-0.5 ml centrifugal filters with a cut-off of 3K and separated on 12% SDS-PAGE as described by Ortega Ramirez et al 2018 and analyzed on a nano liquid chromatography-ion trap mass spectrometer connected to a C18 column. Spectral counts were obtained using the Myrimatch search engine algorithm (Tabb et al, 2007) and then with IdPicker (Ma et al, 2009). To determine relative protein abundance, log2 (spectral counts) will be normalized using the central tendency of the mean. The relative abundance of protein between activated and inactivated bifidobacterium infantis will be compared for statistical significance. The second part will be extracted with ethyl acetate and dried under a stream of nitrogen, according to Orteag Ramirez et al International Biodeterioration & Biodeteration (2018) 130: 40-47.
To find other signal molecules (e.g., glycolipids or lipids), the experimental samples (activated and inactivated) will be analyzed on MALDI-TOF MS calibrated with malto-oligosaccharides. Using sandwich technique, calibrators and samples were mixed at 2: DHB matrix/sample ratio of 1. The mixture is then spotted on a MALDI target plate. The signal molecules were analyzed in a forward mode as recommended by the instrument manufacturer.
Step 5. measuring the activation of dendritic cells in culture
The same number of cells (activated, not activated) from each group was used. The cells are incubated with a cell culture of dendritic cells. After overnight incubation at 37 ℃, the supernatant was gently removed from the petri dish. The dendritic cells are washed to remove any remaining culture broth and any bifidobacterium infantis not bound to the dendritic cells. MRS was added to the culture dish and the plate was shaken vigorously to displace (place) any bound bifidobacterium infantis. CFU/ml of bifidobacterium infantis in the activated group was compared with the non-activated group. This provides an initial screening step to observe key structures, such as solute binding proteins, that are induced during activation. Dendritic cells were examined for activation pathways, which are known to be important in antigen recognition, including pattern recognition, receptor expression, cytokine production, MHC class II, and DECTIN-1. In other experiments, dendritic cells were co-cultured overnight with activated bifidobacterium infantis in the presence of anti-inflammatory cytokines including IL-10 before their isolation. The treated dendritic cells were then co-cultured with naive T cells in the presence of retinoic acid at 37 ℃ overnight. Specific markers characterizing T cell differentiation include Treg markers contained elsewhere.
In another set of experiments, bifidobacterium infantis cells will be grown for 48 hours on agar plates without yeast extract of MRS comprising lactose (non-activated cells) or human milk oligosaccharides (activated cells) as sole carbon source. The optical density was measured to ensure that the number of cells in the mixture was equal. Exopolysaccharide was gently removed from one culture tube by resuspending the cells in phosphate buffer containing ribonuclease and deoxyribonuclease (1. mu.g/mL and 5. mu.g/mL, respectively) for 1 hour. All cells were harvested by centrifugation (20,000Xg, 4 ℃, 10 min) and resuspended in chemically defined fresh medium (e.g., RPMI medium). The exopolysaccharide-free cells were added to the culture of dendritic cells to determine the level of antigen recognition compared to cells with exopolysaccharides.
Step 6. genomic analysis
Standardized nucleic acid extraction and sequencing Methods are used, for example, Garber et al 2001, Nature Methods, 8: 469, the transcriptome (i.e. the full range of messenger rna (mrna) molecules expressed by activated EPS producing bifidobacterium infantis) was compared with the transcriptome of non-activated non-EPS producing bifidobacterium infantis. The biochemical pathways for extracellular polysaccharide production in activated cells were then elucidated by interpreting transcriptome data using metabolic pathway prediction tools, e.g., Kamburov et al, 2011 Bioinformatics, 27: 2917. the data provided to the prediction algorithm can be further enhanced by generating and integrating metabolomics and/or proteomics data.
Example 14
Isolation of Bifidobacterium infantis cell wall as a component of increasing TREG cell production
Food grade methods are used to produce stable cell wall fragments, including SBP and/or exopolysaccharides, from bifidobacterium infantis. Bifidobacterium infantis was grown on activators (Immunity, see International patent publication No. WO 2016/065324) to a yield of at least 1000 hundred million CFU/ml. Cells were harvested and the supernatant removed. The solution containing the cells is lysed using sonication techniques to disrupt the cells. The mixture was acidified and heated to 35 ℃ to precipitate the membrane and separate it from the remaining lysed cell debris.
The precipitated membrane fraction was added to oil and fed to naive mice for 7 days with vitamin a and the induction of TReg cells was analyzed by isolation of leukocytes for PBMC characterization and assessment of regulatory T cell population expansion using flow cytometry specific for CD4, CD25, Foxp3, Helios, neuropilin, CD8, CD44, CD62L, IFN γ, IL-17.
Mice fed the bifidobacterium infantis composition showed increased levels of white blood cells and T cell populations.
Example 15
Bifidobacterium infantis metabolites comprising human intestinal epithelial cells protected from pathogen induced inflammation
Little is known about the anti-inflammatory effect of bifidobacterium infantis metabolites on intestinal epithelial cells and how they protect and maintain mucosal integrity in the infant gut. To investigate whether metabolites from bifidobacterium infantis grown on HMOs and synthetic HMOs could provide protection against pathogen-induced inflammation and maintain mucosal integrity compared to other common strains that have been characterized previously, bifidobacterium infantis (b.infarnatis), bifidobacterium breve (b.breve), bifidobacterium bifidum (b.bifidum) and bifidobacterium longum (b.longum) were grown in media containing pooled HMOs, synthetic lacto-N-neotetraose (LNnT) or fructo-oligosaccharide (FOS, a readily available oligosaccharide commonly used in children's formulas). 4 Lactobacillus plantarum (Lactobacillus plantarum) strains were also grown in HMO, LNnT and FOS medium. After 48 hours of growth (when the bacteria reached the stationary phase), the used supernatant was collected and filtered and the remaining oligosaccharide concentration was evaluated. Human intestinal epithelial cells (IEC; HT-29) were grown to confluency in 96-well plates, then exposed to cell culture medium containing 15% of the bacterial supernatant used at 37 ℃ for 1 hour, then the medium was removed and cultured with a medium containing the supernatant from E.coli O111: the culture medium of Lipopolysaccharide (LPS) of B4 challenged IEC monolayers. After overnight incubation, cell supernatants were analyzed for reduction of proinflammatory cytokines (including IL-8 and TNF-. alpha.) by ELISA. The amount of mucus produced was compared between groups.
First, the growth curves show that bifidobacterium infantis has a selective growth advantage when grown in HMO compared to other bifidobacterium and lactobacillus strains. These data further indicate that various strains of bifidobacteria and lactobacilli grew well using FOS alone as a carbon source. Furthermore, HPLC data confirm that the growth advantage of bifidobacterium infantis is due to its ability to utilize HMO as a carbon source, since very low concentrations of pooled and synthesized HMO can be measured in the supernatant used. In contrast, high concentrations of pooled and synthetic HMO remained in other strains of bifidobacterium and lactobacillus strains, confirming that HMO provided selective growth for bifidobacterium infantis. All tested strains were able to readily use FOS as a carbon source. Furthermore, 1 hour IEC exposure to the supernatants used from pooled and synthetic MHO or FOS from bifidobacterium infantis grown on pooled and synthetic MHO or FOS significantly reduced the pro-inflammatory response compared to the media alone (P ═ 0.015, 0.01, and 0.0005, respectively). Furthermore, this protective effect is also specific for bifidobacterium infantis compared to other bifidobacterium and specific lactobacillus strains used in this study.
These data indicate that metabolites produced by bifidobacterium infantis provide direct protection against pathogen-induced inflammation in the intestinal mucosa specific to this bacterial strain.
Example 16
Immunological development of infants born by caesarean section
Approximately 25 pairs of mother-infant dichotomies were recruited to provide cord blood samples and two-infant blood samples, one on days 0-4 and the other at 3 months of age (84-104). Markers of immune function were analyzed and compared to the development of the gut microbiome.
The experimental design is illustrated in fig. 16. Changes in immune cell and marker levels from baseline (day 0-4) to 3 months (day 84-104) will be measured. Correlations between gut microbiota composition and abundance and levels of immune cells and markers can also be measured. Differences in immune cell and marker levels between bifidobacterium infantis and placebo supplements in breast-fed infants.
Differences in vaccine response (antibody titers) between bifidobacterium infantis and placebo supplements. Blood samples were collected at baseline and 3 months post-partum for evaluation of peripheral blood mononuclear cell characteristics (including regulatory T cells) and vaccine response.
The effect of the supplement on colic, diaper rash and sleep will be evaluated. Reduced inflammation, increased Treg, decreased levels of Th17 and IL-17, improved diaper rash and/or colic are expected.
This example provides an illustration of a study design that can assess the effect of any composition on the immune system of infants born by caesarean section or other groups in need of correction of dysbiosis. One skilled in the art will recognize that compositions with and without vitamin a or D, or compositions with different types of OS, can be delivered and evaluated using this or similar study design.
All publications, patents, and published patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or published patent application was specifically and individually indicated to be incorporated herein by reference.
PCT/RO/134 Table
Figure QDA0002927731820000011

Claims (158)

1. A composition comprising vitamin a or a vitamin a derivative or metabolite thereof, and an Oligosaccharide (OS).
2. The composition of claim 1, wherein the vitamin a is retinol, retinal, retinoic acid, provitamin a carotenoid, or a combination thereof.
3. A composition as in claim 1 or 2 wherein the provitamin a carotenoid is alpha-carotene, beta-carotene, gamma-carotene, lutein beta-cryptoxanthin, or a combination thereof.
4. A composition according to any one of claims 1-3, wherein the provitamin a carotenoid is β -carotene.
5. The composition of any one of claims 1-4, wherein the composition comprises 1x10,000 international units of vitamin A delivered per day.
6. The composition of any one of claims 1-4, wherein the composition comprises 1x 2,000 international units of vitamin A delivered per day.
7. The composition of claim 4 or 5, wherein the composition comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μmol/l vitamin A.
8. The composition of claim 4 or 5, wherein the composition comprises about 1-100, 5-50, 25-75, 10-100, 30-60, or 75-100 μmol/l vitamin a.
9. The composition of any one of claims 1-8, wherein said Oligosaccharide (OS) comprises one or more oligosaccharides having 2-10 residues (DP2-10 oligosaccharides).
10. The composition of any one of claims 1-9, wherein the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO).
11. The composition of claim 10, wherein the Mammalian Milk Oligosaccharide (MMO) comprises oligosaccharide molecules present in Human Milk Oligosaccharide (HMO), Bovine Milk Oligosaccharide (BMO), Bovine Colostrum Oligosaccharide (BCO), Goat Milk Oligosaccharide (GMO), or a combination thereof.
12. The method of any one of claims 1-11, wherein the mammalian lactooligosaccharide (MMO) comprises lacto-N-disaccharide, N-acetyllactosamide, lacto-N-trisaccharide, N-acetyllactosamide, lacto-N-neotetraose, lacto-N-tetraose, fucosyllactose, lacto-N-fucopentose, lactodifucotetraose, sialyllactose, disialolactone-N-tetraose, 2 '-fucosyllactose, 3' -sialyllactosamine, 3 '-fucosyllactose, 3' -sialyllactose, 6 '-sialyllactosamine, 6' -sialyllactose, difucosyllactose, lacto-N-fucosylpentose I, milk-N-fucosyl pentose II, milk-N-fucosyl pentose III, milk-N-fucosyl pentose V, sialylmilk-N-tetraose, derivatives thereof, or combinations thereof.
13. The composition of any one of claims 1-12, wherein the Mammalian Milk Oligosaccharide (MMO) comprises lacto-N-disaccharide or N-acetyl lactosamide.
14. The composition of any one of claims 1-12, wherein the Mammalian Milk Oligosaccharide (MMO) comprises lacto-N-trisaccharide (LNT) or lacto-N-neotetraose (LNnT).
15. The composition of any one of claims 1-14, wherein at least one of the oligosaccharides has a type I or type II core, or at least one of each.
16. A composition according to any one of claims 1 to 14, wherein the oligosaccharide is derived from a plant, fungus, animal, insect or crustacean.
17. The composition of claim 16, wherein the oligosaccharide is from carrot, pea, broccoli, onion, tomato, pepper, rice, wheat, oat, bran, orange, cocoa, olive, apple, grape, beet, cabbage, corn, shrimp, mushroom, or mixtures thereof.
18. The composition of claim 16 or 17, wherein the oligosaccharide is a pre-digested polysaccharide from orange peel, shrimp, mushroom, cocoa shell, olive pomace, tomato pomace, grape pomace, corn silage or a mixture thereof.
19. The composition of any one of claims 16-18, wherein the plant-derived oligosaccharide is from 2-10 sugar residues (DP2-DP10), from 3-10 sugar residues (DP3-DP10), from 5-10 sugar residues (DP5-DP10), or up to DP 20.
20. The composition of any one of claims 1-19, wherein the oligosaccharide comprises galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), or xylo-oligosaccharide (XOS).
21. The composition of any one of claims 1-20, wherein the Oligosaccharide (OS) comprises a Human Milk Oligosaccharide (HMO).
22. The composition of any one of claims 1-21, wherein the composition provides a total dietary intake of oligosaccharides in an amount of 0.001-100 g/day.
23. The composition of any one of claims 1-22, wherein the amount of oligosaccharide is 1-20 grams, 3-20 grams, or 5-10 grams per unit dose.
24. The composition of any one of claims 1-23, wherein the amount of oligosaccharide is 10, 15, 20, 25, 30, 35, 40, 45, or 50 grams.
25. The composition of any one of claims 1-24, wherein the composition further comprises a Bifidobacterium (Bifidobacterium).
26. The composition of claim 25, wherein the Bifidobacterium is Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium animalis subsp.
27. The composition of claim 25 or 26, wherein the composition comprises activated bifidobacteria.
28. The composition of claim 27, wherein the bifidobacterium longum is bifidobacterium longum subsp.
29. The composition of any one of claims 25-28, wherein the bifidobacterium longum subspecies infantis is an activated bifidobacterium longum subspecies infantis.
30. The composition of claim 29, wherein the bifidobacterium infantis has a functional cluster of H5.
31. The composition of claim 29, wherein the extracellular polysaccharide and solute-binding protein on the cell surface of bifidobacterium infantis is increased.
32. The composition of any one of claims 25-27, wherein the bifidobacterium is bifidobacterium breve.
33. The composition of claim 32, wherein the bifidobacterium breve is activated bifidobacterium breve.
34. The composition of any one of claims 25-33, wherein the composition comprises: bifidobacterium in an amount of 1 to 5000 million Colony Forming Units (CFU) per gram of the composition.
35. The composition of any one of claims 25-33, wherein the composition comprises: bifidobacterium in an amount of from 10 to 1000 million Colony Forming Units (CFU) or from 50 to 200 million Colony Forming Units (CFU) per gram of the composition.
36. The composition of any one of claims 25-33, wherein the amount of bifidobacteria is 10, 50, 150, 200, 250, 300, 350, 400, 450, or 500 million Colony Forming Units (CFU) per gram of composition.
37. The composition of any one of claims 1-36, wherein the composition further comprises an isolated bifidobacterium infantis activated cell membrane comprising exopolysaccharides and/or solute-binding proteins.
38. The composition of any one of claims 1-37, wherein the composition is in the form of a dry powder or a dry powder suspended in an oil.
39. The composition of claim 38, wherein the composition is in the form of a dry powder.
40. The composition of claim 38, wherein the composition is in a form suspended in oil.
41. The composition of any one of claims 1-40, wherein the composition is spray dried or freeze dried.
42. The composition of claim 41, wherein the composition is freeze-dried in the presence of a cryoprotectant.
43. The composition of any one of claims 1-42, wherein the composition further comprises a stabilizer.
44. The composition of claim 43, wherein the stabilizing agent is a flow agent.
45. The composition of claim 44, wherein the stabilizer is a cryoprotectant.
46. The composition of claim 45, wherein the cryoprotectant is glucose, lactose, raffinose, sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethylene glycol, propylene glycol, ribitol, alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidone, taurine, mammalian lactooligosaccharides, polysaccharides, or a combination thereof.
47. The composition of any one of claims 1-46, wherein the composition is: pharmaceutical composition, dietary supplement, nutritional product, food product, probiotic and/or prebiotic.
48. The composition of any one of claims 1-47, wherein the composition is formulated as a unit dose drug.
49. The composition of any one of claims 1 to 48, wherein the composition is formulated as a capsule, packet, sachet, food, lozenge, tablet, optionally effervescent tablet, enema, suppository, dry powder suspended in oil, chewable composition, syrup or gel.
50. The composition of any one of claims 1-49, wherein the composition further comprises an intact protein source or a breakdown product enriched in threonine, N-acetyl-threonine, gamma-glutamyl threonine, or a combination thereof.
51. A nutritional product comprising the composition of any one of claims 1-50.
52. The nutritional product according to claim 51, wherein said product is a food product, a dietary supplement, an infant formula or a pharmaceutical product.
53. A method of preventing and/or treating an autoimmune disease comprising administering the composition of any one of claims 1-52.
54. A method of elevating regulatory T cells (tregs) and/or B cells comprising administering to a subject retinoic acid or a source thereof, an Oligosaccharide (OS) and optionally bifidobacterium.
55. A method of preventing and/or treating an autoimmune disease, comprising administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally bifidobacterium.
56. A method of preventing and/or treating allergy, comprising administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally bifidobacterium.
57. A method for increasing the efficiency of antigen recognition in an animal comprising administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, an Oligosaccharide (OS) and optionally bifidobacterium.
58. The method of claim 56, wherein the efficiency of gene therapy and/or vaccine is increased in a subject in need thereof.
59. A method for maintaining gut mucosal integrity during chemotherapy comprising administering to a subject vitamin a or a vitamin a derivative or metabolite or source thereof, and an Oligosaccharide (OS), optionally, bifidobacterium.
60. A method for preventing and/or treating an autoimmune disease comprising administering:
(a) vitamin a or a vitamin a derivative or metabolite or source thereof;
(b) oligosaccharides (OS); and
(c) bifidobacterium bacteria.
61. A method for preventing and/or treating allergy, comprising administering:
(a) vitamin a or a vitamin a derivative or metabolite or source thereof;
(b) oligosaccharides (OS); and
(c) bifidobacterium bacteria.
62. A method for protecting intestinal barrier integrity during chemotherapy or radiation therapy comprising administering:
(a) oligosaccharides (OS);
(b) a bifidobacterium; and
(c) proteins rich in threonine and/or threonine, N-acetyl threonine and/or gamma-glutamyl threonine;
(d) and optionally, vitamin a or a derivative thereof.
63. A method for maintaining gut mucosal integrity during chemotherapy comprising administering:
(a) vitamin a or a vitamin a derivative or metabolite or source thereof;
(b) oligosaccharides (OS);
(c) a bifidobacterium; and
(d) proteins rich in threonine and/or threonine, N-acetyl threonine and/or gamma-glutamyl threonine.
64. A method for stimulating regulatory t (treg) cells comprising administering:
(a) oligosaccharides (OS);
(b) a bifidobacterium; and
(c) optionally, vitamin a or a derivative thereof.
65. A method for stimulating mucin production comprising administering:
(a) vitamin a or a vitamin a derivative or metabolite or source thereof;
(b) oligosaccharides (OS);
(c) a bifidobacterium; and
(d) optionally threonine and/or threonine, N-acetyl threonine and/or gamma-glutamyl threonine rich proteins.
66. The method of claim 55 or 56 or 61, wherein the autoimmune disease is inflammatory bowel disease or celiac disease.
67. The method of claim 66, wherein the Inflammatory Bowel Disease (IBD) is Ulcerative Colitis (UC) or Crohn's disease.
68. The method of any one of claims 53-67, wherein the subject has a high inflammatory bowel.
69. The method of claim 56 or 61, wherein the allergy is food allergy or atopy.
70. The method of any one of claims 53-69, wherein the subject is a mammal.
71. The method of claim 69, wherein the mammal is a human, cow, pig, rabbit, goat, sheep, cat, dog, horse, llama, or camel.
72. The method of claim 70 or 71, wherein the mammal is an infant.
73. The method of claim 72, wherein said mammal is a lactating infant mammal.
74. The method of any one of claims 53-73, wherein the subject is a human.
75. The method of any one of claims 53-74, wherein the vitamin A is retinol, retinal, retinoic acid, provitamin A carotenoid, or a combination thereof.
76. The method of claim 75, wherein the provitamin A carotenoid is alpha-carotene, beta-carotene, gamma-carotene, lutein beta-cryptoxanthin, or a combination thereof.
77. A process as in claim 75 or 76 wherein said provitamin A carotenoid is β -carotene.
78. The method of any one of claims 53-77, wherein said composition comprises 1-100 μmol/l vitamin A.
79. The method of any one of claims 53-78, wherein the composition comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μmol/l vitamin A.
80. The method of any one of claims 53-78, wherein the composition comprises about 1-100, 5-50, 25-75, 10-100, 30-60, or 75-100 μmol/l vitamin A.
81. The method of any one of claims 53 to 80, wherein said Oligosaccharide (OS) comprises DP3-10 oligosaccharide in one or more mammalian milks, said DP3-10 oligosaccharide having a structure of 3-10 sugar residues.
82. The method of claim 81, wherein the oligosaccharide is a Mammalian Milk Oligosaccharide (MMO).
83. The method of claim 82, wherein the Mammalian Milk Oligosaccharide (MMO) comprises a Human Milk Oligosaccharide (HMO), a Bovine Milk Oligosaccharide (BMO), a Bovine Colostrum Oligosaccharide (BCO), a Goat Milk Oligosaccharide (GMO), or a combination thereof.
84. The method of claim 82 or 83, wherein the Mammalian Milk Oligosaccharide (MMO) comprises one or more selected from the group consisting of: lacto-N-disaccharide, N-acetyllactosamide, lacto-N-trisaccharide, lacto-N-neotetraose, lacto-N-tetraose, fucosyllactose, lacto-N-fucopentaose, lacto-difucotetraose, sialyllactose, disialolactone-N-tetraose, 2' -fucosyllactose, 3' -sialyllactosamine, 3' -fucosyllactose, 3' -sialyl-3-fucosyllactose, 3' -sialyllactose, 6' -sialyllactosamine, 6' -sialyllactose, difucosyllactose, lacto-N-fucosylpentaose I, lacto-N-fucosylpentaose II, lacto-N-fucosylpentaose III, lacto-N-fucosylpentaose V, sialyllacto-N-tetraose, derivatives thereof or combinations thereof.
85. A method according to any one of claims 53 to 84, wherein the oligosaccharide is derived from an oligosaccharide of fungal, insect, crustacean or plant origin, optionally predigested from a source polysaccharide.
86. The method of claim 85, wherein the oligosaccharide is from carrot, pea, broccoli, onion, tomato, chili, rice, wheat, oat, bran, orange, cocoa, olive, apple, grape, beet, cabbage, corn, soybean, shrimp, mushroom, or a mixture thereof.
87. The method of claim 85 or 86, wherein the plant oligosaccharide is from orange peel, cocoa pod, olive pomace, tomato pomace, grape pomace, corn silage, or a mixture thereof.
88. The method of claim 87, wherein the plant-derived oligosaccharide is from 2 to 10 sugar residues (DP2-DP10), from 3 to 10 sugar residues (DP3-DP10), from 5 to 10 sugar residues (DP5-DP10), or greater than DP 30.
89. The method of any one of claims 53-88, wherein the oligosaccharide is a galacto-oligosaccharide (GOS), a fructo-oligosaccharide (FOS), or a xylo-oligosaccharide (XOS).
90. The method of any one of claims 53-89, wherein the Oligosaccharide (OS) comprises a Human Milk Oligosaccharide (HMO).
91. The method of any one of claims 53-90, wherein said composition further comprises galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), or xylo-oligosaccharides (XOS).
92. The method of any one of claims 53-91, wherein the oligosaccharide comprises at least one type II core.
93. The method of any one of claims 53-91, wherein the oligosaccharide comprises at least one type I core.
94. The method of any of claims 53-93, wherein the composition provides a total daily oligosaccharide intake in an amount of 0.1-50 g/day, regardless of delivery form or dosing regimen.
95. The method of any one of claims 53-94, wherein the amount of oligosaccharide is 1-20 g, 3-20 g, or 5-10 g.
96. The method of any one of claims 53-95, wherein the amount of oligosaccharide is 10, 15, 20, 25, 30, 35, 40, 45, or 50 grams.
97. The method of any one of claims 53-96, wherein the Bifidobacterium is Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium animalis subsp.
98. The method of claim 97, wherein the bifidobacterium is bifidobacterium longum subsp.
99. The method of claim 98, wherein the bifidobacterium longum subspecies infantis is an activated bifidobacterium longum subspecies infantis.
100. The method of any one of claims 53-99, wherein the bifidobacterium is bifidobacterium breve.
101. The method of claim 100, wherein the bifidobacterium breve is activated bifidobacterium breve.
102. The method of any one of claims 53 to 101, wherein the amount of Bifidobacterium is in the range of from 1 to 5000 million Colony Forming Units (CFU) per gram of composition.
103. The method of any one of claims 53 to 102, wherein the amount of bifidobacteria is from 10 to 1000 million Colony Forming Units (CFU) or from 50 to 200 million Colony Forming Units (CFU) per gram of composition or colony forming units per μ g DNA.
104. The method of any one of claims 53-103, wherein the Bifidobacterium is present in an amount of 10, 50, 150, 200, 250, 300, 350, 400, 450 or 500 million Colony Forming Units (CFU) per gram of composition.
105. The method of any one of claims 53-104, wherein said vitamin A or vitamin A derivative or metabolite or source thereof, Oligosaccharide (OS), Bifidobacterium, or a combination thereof is in a composition.
106. The method of claim 105, wherein the composition is in the form of a dry powder or a dry powder suspended in an oil.
107. The method of claim 105, wherein the composition is spray dried or freeze dried.
108. The method of claim 105, wherein the composition is lyophilized in the presence of a cryoprotectant.
109. The method of claim 108, wherein the composition further comprises a stabilizer.
110. The method of claim 109, wherein the stabilizing agent is a flow agent.
111. The method of claim 110, wherein the stabilizing agent is a cryoprotectant.
112. The method of claim 111, wherein the cryoprotectant is glucose, lactose, raffinose, sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethylene glycol, propylene glycol, ribitol, alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidone, taurine, mammalian lactooligosaccharides, polysaccharides, or a combination thereof.
113. The method of any one of claims 53-112, wherein the composition is formulated as a unit dose drug.
114. The method of any one of claims 53-113, wherein the composition is: pharmaceutical composition, dietary supplement, nutritional product, food product, probiotic and/or prebiotic.
115. The method of any of claims 53-114, wherein the composition is formulated as a capsule, packet, sachet, food, lozenge, tablet, optionally effervescent tablet, enema, suppository, dry powder suspended in oil, chewable composition, syrup or gel.
116. The method of any one of claims 53-115, wherein the Oligosaccharides (OS) constitute at least about 15%, at least 25%, at least 50%, at least 75%, at least 95% of the subject's total dietary fiber.
117. The method of any one of claims 53-116, wherein elevating regulatory T cells (Tregs) results in the suppression of toxic T helper (Th) cells.
118. The method of any one of claims 53-117, wherein elevating regulatory T cells (Tregs) results in a decrease in inflammatory markers.
119. The method of any one of claims 53-118, wherein the inflammatory marker is IL-8, IL-6, TNF-a, IL-10INF γ, INF α, or a combination thereof.
120. The method of claim 118 or 119, wherein the inflammatory marker is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
121. The method of any one of claims 53-120, wherein the subject has been colonized with a bifidobacterium species as measured by bifidobacterium species CFU/gram stool or CFU/μ gDNA.
122. The method of any one of claims 53-121, wherein the amount of bifidobacteria species colonization in the subject is increased by at least 1-10CFU per gram of fecal matter.
123. The method of any one of claims 53-122, wherein the subject is not colonized by a bifidobacterium species as measured by bifidobacterium species CFU per gram of stool.
124. The method of any of claims 53-123, wherein the dose of retinoic acid or a source thereof, Oligosaccharide (OS), Bifidobacterium, or a combination thereof is in an amount effective to maintain a Bifidobacterium level of at least 106At least 108CFU/g feces or 108CFU/. mu.g DNA, or the relative abundance of bifidobacteria in the microbiome is at least 10%, at least 20%, at least 30%At least 50%, at least 60%, at least 70%, at least 80% or at least 90%, or by BLON2175, BLON2175 and/or BLON2177 gene abundance.
125. The method of any one of claims 53-124, wherein the bifidobacterium is administered to the subject daily comprising 1 to 5000 hundred million CFU bacteria per day.
126. The method of any one of claims 53 to 125, wherein the bifidobacteria are administered daily and may comprise from 10 to 1000 or 50 to 200 million CFU per day.
127. The method of any one of claims 53-126, wherein the Bifidobacterium is administered daily for at least 1,2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 to 365 days.
128. The method of any one of claims 52-126, wherein the bifidobacterium is administered daily for about 1-5, 6-10, 11-15, 16-20, 21-25, or 26-30 days.
129. The method of any one of claims 53-128, wherein the oligosaccharide is administered in solid or liquid form.
130. The method of any of claims 53-129, wherein the oligosaccharide is administered in an amount of from about 0.1-50 g/day.
131. The method of any one of claims 53-130, wherein the oligosaccharide is administered in an amount of from about 2-30 g/day or 3-10 g/day.
132. The method of any one of claims 53-131, wherein a first composition comprising retinoic acid and oligosaccharide is administered to the subject.
133. The method of claim 132, wherein the first composition is administered multiple times a day, optionally 1-6 times a day.
134. The method of claim 132 or 133, wherein the first composition is administered for at least 1-365 days.
135. The method of any one of claims 53-134, wherein a second composition comprising bifidobacteria is administered to the subject.
136. The method of claim 135, wherein the second composition is administered daily.
137. The method of claim 135 or 136, wherein the second composition is administered for at least 1-365 days.
138. The method of any one of claims 53 to 137, wherein said first composition comprising retinoic acid or a source thereof and oligosaccharides is administered to said subject prior to administration of said second composition comprising bifidobacteria.
139. The method of any of claims 53-138, wherein a third composition comprising retinoic acid, oligosaccharides and bifidobacteria is administered to the subject.
140. The method of any of claims 53-139, wherein said vitamin A or vitamin A derivative or metabolite or source thereof is administered multiple times a day for at least 1-30 days.
141. The method of any one of claims 53-140, wherein the oligosaccharide is administered multiple times a day for at least 1-30 days.
142. The method of any one of claims 53-141, wherein the Bifidobacterium is administered daily for at least 1-30 days.
143. The method of any of claims 53-142, wherein the vitamin A or vitamin A derivative or metabolite or source thereof, oligosaccharides and bifidobacteria are administered to the subject daily in a composition for at least 1-30 days.
144. The method of any one of claims 53 to 143, wherein the vitamin A or vitamin A derivative or metabolite or source thereof and oligosaccharide is administered to the subject multiple times daily for at least 1-30 days, followed by administration of Bifidobacterium daily for at least 1-30 days.
145. The method of any one of embodiments 53-144, wherein the function of the immune system is enhanced in the mammal following administration of the bacterium, the MMO, or both.
146. The method of claim 145, wherein the immune system function enhancement is a systemic improvement: vaccine response, mucosal innate or adaptive immunity and/or improving homeostasis of innate and adaptive immunity.
147. The method of claim 146, wherein the immune system function is manifested by: in response to increased antibody titres in the vaccine, mucus production is improved, the population of regulatory T cells and B cells is increased, or the production of sIgA in the gut is increased.
148. The method of claim 146 or 147, wherein the increase is statistically significant.
149. The method of any one of claims 144-148, wherein the increase is about 20, 30, 40, 50, 60, 70, 80, or 90%.
150. The method of any one of claims 53-149, wherein the method further comprises administering a threonine and/or threonine, N-acetyl threonine and/or γ -glutamyl threonine rich protein.
151. A composition for treating Inflammatory Bowel Disease (IBD) comprising the composition of any of claims 1-51.
152. A composition for elevating regulatory T cells (Tregs) comprising the composition of any one of claims 1-51.
153. A composition for preventing and/or treating an autoimmune disease, comprising the composition of any one of claims 1-51.
154. The composition of claim 153, wherein the autoimmune disease is asthma, atopy, or type I diabetes.
155. A composition for preventing and/or treating allergy, comprising the composition of any one of claims 1-51.
156. The composition of claim 154, wherein the autoimmune disease is asthma, atopy, or type I diabetes.
157. A composition for preventing and/or treating cancer, comprising the composition of any one of claims 1-51.
158. A composition for maintaining gut mucosal integrity during chemotherapy comprising the composition of any one of claims 1-51.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220233561A1 (en) * 2016-06-05 2022-07-28 Glycom A/S Human milk oligosaccharides for use in the treatment of symptoms in a patient suffering from non-coeliac wheat and / or gluten sensitivity
US11872231B2 (en) 2019-12-09 2024-01-16 Nicoventures Trading Limited Moist oral product comprising an active ingredient
US11969502B2 (en) 2019-12-09 2024-04-30 Nicoventures Trading Limited Oral products
WO2022091075A1 (en) * 2020-11-01 2022-05-05 Aili Life Sciences Ltd. Combination compositions of probiotics with fermented wheat germ extract and uses thereof
US20240009216A1 (en) * 2020-11-10 2024-01-11 Societe Des Produits Nestle S.A. Nutritional composition
TW202302128A (en) * 2021-03-26 2023-01-16 香港中文大學 Use of microorganisms to improve vaccine efficacy

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080118473A1 (en) * 2006-11-01 2008-05-22 The Procter & Gamble Company Methods of treating a respiratory condition comprising probiotic treatment
CN102089422A (en) * 2008-07-11 2011-06-08 科.汉森有限公司 New probiotic bifidobacterium longum
US20120202753A1 (en) * 2009-07-06 2012-08-09 Children's Hospital Medical Center D/B/A Cincinnati Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
CN103458890A (en) * 2011-04-12 2013-12-18 雀巢产品技术援助有限公司 Nutritional compositions including branched chain fatty acids and methods of using same
CN103763940A (en) * 2010-12-31 2014-04-30 雅培制药有限公司 Synbiotic combination of probiotic and human milk oligosaccharides to promote growth of beneficial microbiota
US20150342974A1 (en) * 2012-12-18 2015-12-03 Abbott Laboratories Nutritional compositions comprising neuroprotective dietary oligosaccharides
CN107109348A (en) * 2014-08-29 2017-08-29 科.汉森有限公司 Probiotics bifidobacterium adolescentis bacterial strain
CN107249611A (en) * 2014-10-24 2017-10-13 进化生物系统股份有限公司 The Bifidobacterium of activation and its application process

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1993577B1 (en) * 2006-03-10 2014-02-12 N.V. Nutricia Use of non-digestable sacharides for giving an infant the best start after birth
AU2016209244A1 (en) * 2015-01-23 2017-08-17 Helena M.G.P.V. Reis Use of short chain fatty acids in cancer prevention
EP3419630A4 (en) * 2016-02-24 2019-11-06 Glycom A/S Synthetic composition for microbiota modulation

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080118473A1 (en) * 2006-11-01 2008-05-22 The Procter & Gamble Company Methods of treating a respiratory condition comprising probiotic treatment
CN102089422A (en) * 2008-07-11 2011-06-08 科.汉森有限公司 New probiotic bifidobacterium longum
US20120202753A1 (en) * 2009-07-06 2012-08-09 Children's Hospital Medical Center D/B/A Cincinnati Children's Hospital Medical Center Inhibiting inflammation with milk oligosaccharides
CN103763940A (en) * 2010-12-31 2014-04-30 雅培制药有限公司 Synbiotic combination of probiotic and human milk oligosaccharides to promote growth of beneficial microbiota
CN103458890A (en) * 2011-04-12 2013-12-18 雀巢产品技术援助有限公司 Nutritional compositions including branched chain fatty acids and methods of using same
US20150342974A1 (en) * 2012-12-18 2015-12-03 Abbott Laboratories Nutritional compositions comprising neuroprotective dietary oligosaccharides
CN107109348A (en) * 2014-08-29 2017-08-29 科.汉森有限公司 Probiotics bifidobacterium adolescentis bacterial strain
CN107249611A (en) * 2014-10-24 2017-10-13 进化生物系统股份有限公司 The Bifidobacterium of activation and its application process

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